Skin conditioning compositions containing compounds for mimicking the effect on skin of retinoic acid

ABSTRACT

A skin care product comprising from about 0.001% to about 10% of a retinoid, in combination with 0.0001% to about 50% of a combination of retinoid boosters.

The present invention relates to cosmetic skin conditioning compositionscontaining certain compounds which mimic the effect on skin of retinoicacid.

Retinol (vitamin A) is an endogenous compound which occurs naturally inthe human body, and is essential for normal epithelial celldifferentiation. Natural and synthetic vitamin A derivatives have beenused extensively in the treatment of a variety of skin disorders andhave been used as skin repair or renewal agents. Retinoic acid has beenemployed to treat a variety of skin conditions, e.g., acne, wrinkles,psoriasis, age spots and discoloration. See e.g., Vahlquist, A. et al.,J. Invest. Dermatol., Vol. 94, Holland D. B. and Cunliffe, W. J. (1990),pp. 496-498; Ellis, C. N. et al., “Pharmacology of Retinols in Skin”,Vasel, Karger, Vol. 3, (1989), pp. 249-252; Lowe, N. J. et al.,“Pharmacology of Retinols in Skin”, Vol. 3, (1989), pp. 240-248; PCTPatent Application No. WO 93/19743.

It is believed that retinol esters and retinol are enzymaticallyconverted in the skin into retinoic acid according to the followingmechanism:

The present invention is based on the discovery that certain compoundsenhance the conversion of retinyl esters and retinol to retinoic acid.The compounds are collectively termed “boosters” and are coded as groupsB1 to B5 according to the boosting mechanism of the particular compound.The mechanism of the booster groups is as follows: inhibiting ARAT/LRAT(AcylCoenzymeA (CoA): retinol acyl transferase/Lecithin: retinol acyltransferase) activity (B1), enhancing retinol dehydrogenase activity(B2), inhibiting retinal reductase activity (B3), antagonising CRABP-II(Cellular Retinoic Acid Binding Protein II) binding of retinoic acid(B4) and inhibiting cytochrome P450 dependent retinoic acid oxidation(B5).

The boosters alone or in combination with each other potentiate theaction of retinoids by increasing the conversion of the retinoids toretinoic acid and preventing the degradation of retinoic acid. Theboosters act in conjunction with a retinoid (e.g. retinol, retinylesters, retinal, retinoic acid), the latter being present endogenouslyin the skin. The preferred compositions, however, include a retinoid inthe composition, co-present with a booster or a combination of boosters,to optimise performance.

Several patents by Granger et al describe the use of retinoid boostersin cosmetic compositions to improve the efficacy of retinol and retinylesters (U.S. Pat. Nos. 5,759,556, 5,756,109, 5,747,051, 5,716,627,5,811,110, 5,536,740, 5,747,051, 5,599,548, 5,955,092, 5,885,595,5,759,556, 5,693,330). The boosters described in these patents arerestricted to class B1 and B5. Furthermore Johnson & Johnson have aseries of patents which describe the use of molecules which fall intoclass 5 booster molecules (U.S. Pat. Nos. 5,028,628; 5,037,829;5,151,421; 0,476,852; 5,500,435; 5,583,136; 5,612,354).

The molecules which act as retinoid boosters are common ingredients incosmetic products. There is considerable prior art describing their usein cosmetic compositions. There is substantial prior art describing theuse of two or more of these molecules in the same composition. Some ofthe examples of the prior art are as in U.S. Pat. Nos. 5,665,367,5,747,049, 5,853,705, 5,766,575, and 5,849,310.

However, the prior art does not describe synergy arising fromcombinations of booster molecules. This observation of a synergisticboosting of retinoid activity from combinations of booster molecules wasan unexpected finding. The prior art does not describe optimalconcentrations or ratios of booster molecules or ratios of boostermolecules to that of retinoids. Thus, the present invention is novel inthat by combining cosmetic retinoids with booster molecules, at the mostappropriate concentrations or ratios, a substantial improvement inefficacy of the retinoids is obtained.

The classes of boosters suitable for use in the present inventioninclude but are not limited to the boosters listed in Tables B1 throughto B5.

Best Groups of Boosters

B1 Compounds 1. Fatty Acid Amides These are readily commerciallyavailable and have the added advantage of being surfactants and thushelp generate emulsions suitable for cosmetic preparations. 2. CeramidesThese can additionally act as precursors of stratum corneum barrierceramides. 3. Carotenoids These can offer some UV protection and act asnatural colorants. 4. Flavanoids Natural antioxidants. 5. Cyclicfragrances These are readily commercially available and additionally canbe used to fragrance the product. 6. Non-cyclic These can be used tofragrance the fragrances product. 7. Phospholipid These can be utilisedby skin cells analogues to nourish the generation of barrier components.8. Ureas These are readily commercially available and can also act aspreservatives for the product.

B2 Compounds 1. Phosphatidyl choline Most preferred as most activeactivator of Retinol Dehydrogenase 2. Sphingomyelin

B3 Compounds Arachidonic Acid Fatty Acids which can be Linoleic Aciduseful in maintaining stratum Linolenic Acid corneum barrier MyristicAcid Linoleic Acid Essential Fatty Acids Linolenic Acid Arachidonic AcidNon-essential fatty acids Myristic Acid Glycyrrhetinic Acid Polycyclictriterpene carboxylic acid which is readily obtained from plant sources.Phosphatidyl ethanolamine Can be incorporated into cellular membranes.

B4 Compounds Hexadecanedioic acid Saturated fatty acids.12-hydroxystearic acid Isostearic acid Linseed oil Unsaturated fattyacids Elaidic acid Elaidic acid Solid at room temperature Isostearicacid Hexadecanedioic acid Linseed oil Liquid at room temperature12-hydroxystearic acid

B5 Compounds Bifonazole Antimicotics Climbazole Clotrimazole EconazoleKetoconazole Miconazole Climbazole Readily commercially available LaurylCompounds which are readily hydroxyethylimidazoline commerciallyavailable and have the added advantage of being surfactants and thushelp generate emulsions suitable for cosmetic preparations. QuercetinNaturally occuring flavanoid which has antioxidant properties. CoumarinNatural colorant Quinolines Isoquinolines Metyrapone

The present invention includes, in part, a skin conditioning compositioncontaining from about 0.0001% to about 50%, preferably from 0.001% to10%, most preferably from 0.001% to 5% by weight of the composition of abooster or combination of boosters and a cosmetically acceptablevehicle.

The boosters or combinations thereof included in the inventivecompositions are selected from the group consisting of:

-   -   (a) a booster selected from the group consisting of B2; B3; B4;    -   (b) binary combinations of boosters selected from the group        consisting of:        -   B1/B2; B1/B3; B1/B4; B1/B5; B2/B3, B2/B4; B2/B5, B3/B4;            B3/B5; B4/B5    -   (c) ternary combinations of boosters selected from the group        consisting of:        -   B1/B2/B3; B1/B2/B4; B1/B2/B5; B1/B3/B4; B1/B3/B5; B1/B4/B5;            B2/B3/B4; B2/B3/B5; B2/B4/B5; B3/B4/B5    -   (d) quaternary combinations of boosters selected from the group        consisting of:        -   B1/B2/B3/B4; B1/B2/B3/B5; B1/B2/B4/B5; B1/B3/B4/B5;            B2/B3/B4/B5; and    -   (e) a combination of five groups of boosters:        -   B1/B2/B3/B4/B5.

The preferred compositions include from about 0.001% to about 10%, byweight of the composition of a retinoid.

The compounds included in the present invention as boosters are selectedbased on the ability of such compounds to pass, at a certainconcentration listed in Table A, in-vitro Assays for a specific enzymesas described below under sections 2.1 through to 2.7. Such a booster isincluded in the present invention even if it is not explicitly mentionedherein. Put another way, if a compound inhibits or enhances sufficientlyan enzyme in an assay described below, it will act in combination with aretinoid to mimic the effect on keratinocytes (skin cells) of retinoicacid, and thus it is included within the scope of the present invention.

The term “conditioning” as used herein means prevention and treatment ofdry skin, acne, photo-damaged skin, appearance of wrinkles, age spots,aged skin, increasing stratum corneum flexibility, lightening skincolour, controlling sebum excretion and generally increasing the qualityof skin. The composition may be used to improve skin desquamation andepidermal differentiation.

The presence of the selected compounds in the inventive productsubstantially improves the performance of a retinoid.

The inventive compositions contain, as a preferred ingredient, aretinoid, which is selected from retinyl esters, retinol, retinal andretinoic acid, preferably retinol or retinyl ester. The term “retinol”includes the following isomers of retinol: all-trans-retinol,13-cis-retinol, 11-cis-retinol, 9-cis-retinol, 3,4-didehydro-retinol,3,4-didehydro-13-cis-retinol; 3,4-didehydro-1′-cis-retinol;3,4-didehydro-9-cis-retinol. Preferred isomers are all-trans-retinol,13-cis-retinol, 3,4-didehydro-retinol, 9-cis-retinol. Most preferred isall-trans-retinol, due to its wide commercial availability.

Retinyl ester is an ester of retinol. The term “retinol” has beendefined above. Retinyl esters suitable for use in the present inventionare C₁-C₃₀ esters of retinol, preferably C₂-C₂₀ esters, and mostpreferably C₂, C₃, and C₁₆ esters because they are more commonlyavailable. Examples of retinyl esters include but are not limited to:retinyl palmitater retinyl formate, retinyl acetate, retinyl propionate,retinyl butyrate, retinyl valerate, retinyl isovalerate, retinylhexanoate, retinyl heptanoate, retinyl octanoate, retinyl nonanoate,retinyl decanoate, retinyl undecandate, retinyl laurate, retinyltridecanoate, retinyl myristate, retinyl pentadecanoate, retinylheptadeconoate, retinyl stearate, retinyl isostearate, retinylnonadecanoate, retinyl arachidonate, retinyl behenate, retinyllinoleate, and retinyl oleate.

The preferred ester for use in the present invention is selected fromretinyl palmitate, retinyl acetate and retinyl propionate, because theseare the most commercially available and therefore the cheapest. Retinyllinoleate and retinyl oleate are also preferred due to their efficacy.

Retinol or retinyl ester is employed in the inventive composition in anamount of from about 0.001% to about 10%, preferably in an amount offrom about 0.01% to about 1%, most preferably in an amount of from about0.01% to about 0.5%.

The essential ingredient of the inventive compositions is a compoundwhich passes in vitro Assays described below in sections 2.1 through to2.7. A compound suitable for use in the present invention inhibits orenhances at a concentration listed in Table A an enzyme to at least abroad % listed in Table A.

SECTION A Identification of Booster Table A Booster Test Concentrationsand % Inhibition/Increase

ARAT/LRAT Assay (To identify B1 boosters) Compound InventionConcentration % Inhibition Broad 100 μM >10% Preferred 100 μM >25% MostPreferred 100 μM >40% Optimum 100 μM >50%

Retinol Dehydrogenase Assay (To identify B2 boosters) Compound InventionConcentration % Increase Broad 100 μM >10% Preferred 100 μM >15% MostPreferred 100 μM >20% Optimum 100 μM >25%

Retinal Reductase Assay (To identify B3 boosters) Compound InventionConcentration % Inhibition Broad 100 μM  >5% Preferred 100 μM >10% MostPreferred 100 μM >20% Optimum 100 μM >35%

CRABPII Antagonist Assay (To identify B4 boosters) Compound:RetinoicInvention acid Ratio % Inhibition Broad 7000:1 >25% Preferred7000:1 >50% Most Preferred  70:1 >25% Optimum  70:1 >50%

Retinoic Acid Oxidation Assay(To identify B5 boosters) CompoundInvention Concentration % Inhibition Broad 100 μM >25% Preferred 100μM >45% Most Preferred 100 μM >70% Optimum 100 μM >80%

The in vitro Microsomal Assays employed for determining the suitabilityof the inclusion of the compound in the inventive compositions are asfollows:

1. Materials

All-trans-retinol, all-trans-retinoic acid, palmitoyl-CoA, dilauroylphosphatidyl choline, NAD, and NADPH were purchased from Sigma ChemicalCompany. Stock solutions of retinoids for the microsomal assays weremade up in HPLC grade acetonitrile. All retinoid standard stocksolutions for HPLC analysis were prepared in ethanol, stored underatmosphere of N₂ at −70° C. and maintained on ice under amber lightingwhen out of storage. Other chemicals and the inhibitors werecommercially available from cosmetic material suppliers or chemicalcompanies such as Aldrich or International Flavours and Fragrances.

2. Methods

2.1 Isolation of RPE Microsomes (Modified from (1))

50 frozen hemisected bovine eyecups, with the retina and aqueous humorremoved were obtained from W. L. Lawson Co., Lincoln, Nebr., USA. Theeyes were thawed overnight and the colored iridescent membrane wasremoved by peeling with forceps. Each eyecup was washed with 2×0.5 mLcold buffer (0.1M PO4/1 mM DTT/0.25M sucrose, pH 7) by rubbing thedarkly pigmented cells with an artist's brush or a rubber policeman. Thecell suspension was added to the iridescent membranes and the suspensionwas stirred for several minutes in a beaker with a Teflon stir bar. Thesuspension was filtered through a coarse filter (Spectra/Por 925μ poresize polyethylene mesh) to remove large particles, and the resultingdarkly colored suspension was homogenized using a Glas-Col with a motordriven Teflon homogenizer.

The cell homogenate was centrifuged for 30 min. at 20,000 g (Sorvaalmodel RC-5B centrifuge with an SS34 rotor in 2.5×10 cm tubes at 14,000RPM). The resulting supernatant was subjected to further centrifugationfor 60 min. at 150,000 g (Beckman model L80 Ultracentrifuge with anSW50.1 rotor in 13×51 mm tubes at 40,000 RPM). The resulting pelletswere dispersed into ˜5 mL 0.1M PO₄/5 mM DTT, pH 7 buffer using a HeatSystems Ultrasonics, Inc. model W185D Sonifier Cell Disruptor, and theresulting microsomal dispersion was aliquoted into small tubes andstored at −70° C. The protein concentrations of the microsomes weredetermined using the BioRad Dye binding assay, using BSA as a standard.

2.2 Isolation of Rat Liver Microsomes (4)

Approximately 6 grams of frozen rat liver (obtained from Harlan SpragueDawley rats from Accurate Chemical and Scientific Corp.) was homogenizedin 3 volumes of 0.1M tris/0.1M KCl/1 mM EDTA/0.25M sucrose, pH 7.4buffer using a Brinkmann Polytron. The resulting tissue suspension wasfurther homogenized in the motor driven Teflon homogenizer describedabove. The resulting homogenate was successively centrifuged for 30 min.at 10,000 g, 30 min. at 20,000 g, and 15 min. at 30,000 g, and theresulting supernatant was ultra-centrifuged for 80 min. at 105,000 g.The pellet was sonicated in ˜5 mL of 0.1M PO4/0.1 mM EDTA/5 mM MgCl₂, pH7.4 buffer as described above and stored as aliquots at −70° C. Proteinconcentrations were determined as described above.

2.3 Assay for ARAT and LRAT Activity (To Identify B1)

The procedure below was a modification of a method described in theliterature (2). The following buffer was prepared and stored at 4° C.,0.1M PO₄/5 mM dithiothreitol, pH 7.0 (PO₄/DTT). On the day of the assay,2 mg BSA per mL of buffer was added to give a PO₄/DTT/BSA workingbuffer. 1 mM retinol substrate was prepared in acetonitrile and storedin amber bottles under nitrogen gas at −20° C. Solutions of 4 mMPalmitoyl-CoA in working buffer (stored in aliquots) and 4 mM dilauroylphosphatidyl choline in ethanol were prepared and stored at −20° C.Inhibitors were prepared as 10 mM stock solutions in H₂O, ethanol,acetonitrile or DMSO. The quench solution was prepared using pureethanol containing 50 μg/mL butylated hydroxytoluene (BHT), and a hexanesolution containing 50 μg/mL BHT was used for the extractions.

To a 2 dram glass vial, the following were added in order: PO₄/DTT/BSAbuffer to give a total volume of 500 μL, 5 μL acyl donor (4 mMpalmitoyl-CoA and/or dilauroyl phosphatidyl choline), 5 μL inhibitor orsolvent blank (10 mM stock or further dilutions) followed byapproximately 15 μg of RPE microsomal protein (approximately 15 μL of a˜1 mg/mL microsomal protein aliquot). The mixture was incubated for 5min. at 37° C. to equilibrate the reaction temperature and then 5 μL 1mM retinol was added. The vials were capped, vortexed for 5 seconds andincubated for 30-90 minutes at 37° C. The reaction was quenched byadding 0.5 mL ethanol/BHT. The retinoids were extracted by adding 3 mLhexane/BHT, vortexing the tubes for several seconds several times andcentrifuging the tubes at low speed for 5 min. to quickly separate thelayers. The upper hexane layer was removed into a clean vial, and theaqueous layer re-extracted with another 3 mL hexane/BHT, as describedabove. The hexane layers were combined, and the hexane evaporated bydrying at 37° C. under a stream of nitrogen gas on a heated aluminumblock. The dried residue was stored at −20° C. until HPLC analysis. Theamount of retinyl palmitate and retinyl laurate was quantitated for ARATand LRAT activity, respectively, by integration of the HPLC signal asdescribed below.

Note that the incubation solution contains 40 μM acyl donor, 100 μM orless inhibitor, 10 μM retinol, approximately 30 μg/mL microsomalprotein, and nearly 0.1M PO₄/pH 7/5 mM DTT/2 mg/mL BSA. All stepssubsequent to the addition of retinol were done in the dark or underamber lights.

2.4 Assay for Retinol Dehydrogenase Activity (To Identify B2)

The following stock solutions were prepared:

-   50 mM KH2PO4, pH 7.4 buffer, sterile filtered.-   10 mM all trans Retinol (Sigma R7632) in DMSO-   200 mM Nicotinamide adenine dinucleotide phosphate, sodium salt    (NADP) (Sigma N0505) in sterile water.-   40 mM test compound in appropriate solvent (water, buffer, ethanol,    chloroform or DMSO).-   1:10 dilution of rat liver Microsomes in 50 mM KH2PO4, pH 7.4 buffer    (4 μg/μl).

In a two-dram glass vial with screw cap, the following were added inorder:

-   Buffer to give a final volume of 400 μl-   25 μl diluted Microsomes (final=100 μg)—boiled Microsomes were used    for controls and regular Microsomes for test samples.-   4 μl of 200 mM NADP (final=2 mM)-   1 μl of 40 mM test compound (final=100 μM)-   8 μl of 10 mM retinol (final=200 μM)

The vials were incubated in a 37° C. shaking water bath for 45 minutes500 μl ice-cold ethanol was added to each vial to quench the reaction.The retinoids were extracted twice with ice cold hexane (2.7 ml perextraction). Retinyl acetate (5 μl of a 900 μM stock) was added to eachvial during the first extraction as a means of monitoring the extractionefficiency in each sample. Samples were vortexed for ten seconds beforegently centrifuging for five minutes at 1000 rpm, 5° C. in a BeckmanGS-6R centrifuge. The top hexane layer containing the retinoids wasremoved from the aqueous layer after each extraction to a clean two-dramvial. The hexane was evaporated off under a gentle stream of nitrogengas. The dried residue was then stored at −20° C. until HPLC analysis.

2.5 Assay for Retinal Reductase Activity (To identify B3)

All stock solution were prepared as above with the followingsubstitutions:

-   10 mM all trans Retinaldehyde (Sigma R2500) in DMSO-instead of    retinol.-   200 mM, Nicotinamide adenine dinucleotide phosphate, reduced form,    tetrasodium salt (NADPH) (Sigma N7505) in sterile water—instead of    NADP.

In a two-dram glass vial with screw cap, add the following in order:

-   Buffer to give a final volume of 400 μl-   25 μl diluted Microsomes (final=100 μg)—use boiled Microsomes for    controls and regular Microsomes for test samples.-   4 μl of 200 mM NADPH (final=2 mM)-   1 μl of 40 mM test compound (final=100 μM)-   3 μl of 10 mM retinaldehyde (final=75 μM)

Follow the same incubation and extraction procedure as detailed above.

2.6 Assay for CRABPII Antagonists (To Identify B4)

2.6.1. Synthesis of CRABPII

a. System of Expression

The gene CRABPII was cloned in pET 29a-c(+) plasmid (Novagen). Thecloned gene was under control of strong bacteriophage T7 transcriptionand translation signals. The source of T7 polymerase was provided by thehost cell E. coli BLR(DE3)pLysS (Novagen). The latter has a chromosomalcopy of T7 polymerase under lacUV5 control, induced by the presence ofIPTG.

The plasmid was transferred into E. coli BLR(DE3)pLysS cells bytransformation according to the manufacturer protocol (Novagen).

b. Induction

An overnight culture of the transformed cells was diluted 1:100 into2×YT containing 50 μg/mL kanamycin and 25 μg/mL chloramphenicol. Thecells grew while shaking at 37° C. until the OD at 600 nm reached0.6-0.8. Then IPTG was added to a final concentration of 1 mM and theculture was incubated for an additional two hours. The cells wereharvested by centrifugation at 5,000 g for 10 minutes at roomtemperature. The pellet was stored at −20° C.

2.6.2. Purification

Purification was performed according to the method described in Norrisand Li, 1997.

a. Lysis

The frozen pellet was thawed at RT and resuspended in 1-2 pellet volumesof freshly prepared lysis buffer (50 mM Tris-HCl, pH 8, 10% (w/v)sucrose, 1 mM EDTA, 0.05% (w/v) sodium azide, 0.5 mM DTT, 10 mM MnCl₂,2.5 mM phenylmethylsulfonyl fluoride, 2.5 mM benzamidine, 6 μg/mLDNase). The lysate was incubated for 30 mins. at room temperature.Further lysis was accomplished by sonication (six 30-sec bursts at10,000 psi alternated with five 30-sec delay on ice). The insolublefraction of the lysate was removed by centrifugation at 15,000 rpm 1hour at 4° C. and the supernatant is stored at −20° C.

b. Gel Filtration on Sephacryl S300

The supernatant from step a. was loaded onto a 2.5×100 cm column ofsephacryl S-300 (Pharmacia) at room temperature. The elution buffer was20 mM Tris-HCl, pH 8, 0.5 mM DTT, 0.05% sodium azide (buffer A). Theflow rate was 2 mL/min. Collected 2-mL fractions were checked forultraviolet absorbance at 280 nm. The fractions representing the peakswere examined by SDS-page for the presence of CRABPII.

c. Anion-exchange Chromatography.

2 mL of gel filtration fractions containing CRABPII were loaded onto aquaternary amine anion-exchange column FPLC (Fast Protein LiquidChromatography) type monoQ (Pharmacia). CRABPII was eluted using agradient buffer from 100% buffer A to 30% buffer B (100% buffer B=bufferA+250 mM NaCl) over a 20-min period at room temperature. 1 mL-fractionswere collected every minute. Once more, the presence of CRABPII waschecked by SDS page. CRABPII was stored at 4° C. before freeze-dryingusing a Micromodulyo 1.5K with vial platform attachment (Edwards HighVacuum International). The desiccated samples were stored at roomtemperature until their use in the binding assay.

d. Detection of the Presence of CRABPII

The expression and purification of CRABPII was validated usingdenaturing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis ona 7-15% polyacrylamide gel (Biorad). 10 μL samples were mixed with 10 μLof 2× loading buffer (100 mM Tris-HCl pH6.8, 4% SDS, 0.2% BPB, 20%glycerol, 1 mM DTT) and denatured by heating (2 mins. at 80° C.). Thesamples were loaded onto the gel that was immersed in a 1× Tris-glycinebuffer (Biorad) and a constant current (25 mA) was applied for 1 hour atroom temperature. After Coomassie blue staining, the protein wasidentified according to its molecular weight as determined with theBenchmark pre-stained protein ladder (Gibco BRL).

A western blot was used to confirm the presence of CRABPII. The proteinsseparated on the SDS-PAGE were transferred on an Immobilon-P transfermembrane (Millipore) using a Biorad cassette. The transfer occurred in1× Tris-glycine buffer (Biorad)+10% methanol. An electrical currant (60mA) was applied for 3 hours to allow the protein to migrate through themembrane. Afterwards, the membrane was blocked with 5% dry milk in 1×TBSfor one hour at room temperature and probed with primary antibodies toCRABPII (1/1000 dilution of mouse anticlonal 5-CRA-B3) in the samebuffer at 4° C. overnight. The following day, the membrane was washedwith PBS (3×5 minutes) and then incubated with 1:2000 dilution of thesecondary antibody, peroxidase conjugated anti-mouse antibody (ECL™,Amersham), for 1 hour at room temperature. The membrane was washed with1×PBS (3×5 minutes) and the protein was detected using ECL detection kitaccording to the manufacturer instruction (Amersham).

The concentration of purified CRABPII was determined using BSA kit(Pierce).

2.6.3. Radioactive Binding Assay

220 pmol of CRABPII was incubated in 20 mM Tris-HCl buffer pH 7.4 with15 pmol of radioactive all trans retinoic acid (NEN) in a total volumeof 70 μL. For the competitive assay, another ligand in excess (6670:1,670:1 or 70:1) was added to the mix. The reaction occurred for one hourat room temperature in the dark. In order to separate the unboundall-trans retinoic acid from the bound all-trans retinoic acid, a 6 kDcut-off minichromatography column (Biorad) was used. The storage bufferwas discarded using a Microplex manifold for according to themanufacturer instruction (Pharmacia). The samples were loaded onto thecolumn and the separation occurred by gravity over a 30-min period.Retinoic acid (“RA”) bound to CRABPII appeared in the filtrate whilefree RA remained in the column. The radioactivity of the filtrate wasmeasured by scintillation counter.

2.7 Assay for NADPH Dependent Retinoic Acid Oxidation (To Identify B5)

The procedure below is a modification of a method described in theliterature (4). The following assay buffer was prepared and stored at 4°C.: 0.1M PO₄/0.1 mM EDTA/5 mM MgCl₂, pH 7.4. On the day of the assay, a60 mM NADPH solution in buffer was prepared. Inhibitor stocks, acidifiedethanol/BHT quench solution, and hexane/BHT were prepared as describedabove. A working 1 mM retinoic acid solution was prepared by dilution ofa 15 mM stock (in DMSO) with ethanol.

To a 2 dram vial, the following were added in order: assay buffer togive a final volume of 500 μL, 20 μL 60 mM NADPH, 5 μL inhibitor orsolvent blank, followed by approximately 2 mg of rat liver microsomalprotein.

The mixture was incubated for 5 mins. at 37° C., then 5 μL working 1 mMretinoic acid solution was added. Incubation was continued for 60 mins.at 37° C.—the vials were not capped, since the oxidation processrequired molecular O₂ in addition to NADPH. Quenching was carried outwith acidified ethanol/BHT and extraction was carried out withhexane/BHT as described above. Quantitation of the quickly eluting polarretinoic acid metabolites (presumed to be 4-oxo retinoic acid) wascarried out by integration of the HPLC signal as described below.

All steps subsequent to the addition of retinoic acid were done in thedark or under amber lights. The final incubation solution contained 2.4mM NADPH, 100 μM or less inhibitor, 10 μM retinoic acid, approximately 4mg/mL rat liver microsomal protein and nearly 0.1M PO₄/0.1 mM EDTA/5MMgCl₂.

HPLC Analysis of Individual Retinoids

Samples for retinoid quantitation by HPLC were prepared by dissolvingthe residue in each vial with 100 μL of methanol. The solution wastransferred to a 150 μL glass conical tube within a 1 mL shell vial,capped tightly, and placed inside a Waters 715 Autosampler. Aliquots of60 μL were injected immediately and analysed for retinoid content.

The chromatography instrumentation consisted of a Waters 600 gradientcontroller/pump, a Waters 996 Photodiode Array detector and a Waters 474Scanning Fluorescence detector. Two HPLC protocols were used forretinoid analysis. For the ARAT and LRAT assay, the separation ofretinol and retinol esters was performed with a Waters 3.9×300 mm C18Novapak reverse-phase analytical column and Waters Sentry NovaPak C18guard column with an 80:20 (v/v) methanol/THF isocratic mobile phaseadjusted to a flow rate of 1 mL/min. for 10 min. The eluate wasmonitored for absorbance at 325 nm and fluorescence at 325ex/480em.

A shorter Waters 3.9×150 mm C18 Novapak reverse-phase analytical columnand Waters Sentry NovaPak C18 guard column were used to separateretinoid acids and alcohols for the retinol and retinoic acid oxidationassays utilising a modification of a gradient system described by Barua(5). This system consisted of a 20 mins. linear gradient from 68:32(v/v) methanol/water containing 10 mM ammonium acetate to 4:1 (v/v)methanol:dichloromethane followed by a 5 mins. hold at a flow rate of 1mL/min. The column eluate was monitored from 300 nm to 400 nm.

These protocols were selected based on their ability to clearly resolvepertinent retinoid acids, alcohols, aldehydes, and/or esters for eachassay and relative quickness of separation. Identification of individualretinoids by HPLC was based on an exact match of the retention time ofunknown peaks with that of available authentic retinoid standards and UVspectra analysis (300-400 nm) of unknown peaks against availableauthentic retinoids.

REFERENCES

-   1 J. C. Saari & D. L. Bredberg, “CoA and Non-CoA Dependent Retinol    Esterification in Retinal Pigment Epithelium”, J. Bill. Chem. 263,    8084-8090 (1988).-   2 J. C. Saari & D. L. Bredberg, “ARAT & LRAT Activities of Bovine    Retinal Pigment Epithelial Microsomes”, Methods Enzymol. 190,    156-163 (1990).-   3 J. L. Napoli & K. R. Race, “The Biosynthesis of Retinoic Acid from    Retinol by Rat Tissues in vitro”, Archives Biochem. Biophys. 255,    95-101 (1987).-   4 R. Martini & M. Murray, “Participation of P450 3A Enzymes in Rat    Hepatic Microsomal Retinoic Acid 4-Hydroxylation”, Archives Biochem.    Biophys. 303, 57-66 (1993).-   5 A. B. Barua, “Analysis of Water-Soluble Compounds: Glucuronides”,    Methods Enzymol. 189, 136-145 (1990).

The boosters suitable for use in the present invention include but arenot limited to the boosters listed in Tables B₁ through to B₅ below. Thetable below gives the booster class (B₁-B₅), the chemical name of thecompound, and the results from the appropriate assays used to identifythe booster (i.e. ARAT/LRAT for B1, retinol dehydrogenase for B₂,retinaldehyde inhibation for B3, CRABP is binding for B₄ and retinoicacid oxidation inhibition for B₅.

ARAT/LRAT Inhibitors (B1) % Inhibition Overall % Inhibition % Inhibition% Inhibition Overall TG % Inhibition ARAT LRAT LRAT Class CompoundTG(-ROH/RE) (IC 50) ARAT (10jm) (100jm) (10jm) (100jm) CarotenoidCrocetin 3.75E−05 15% 34% 0 15% Fatty Acid & Other Acetyl Sphingosine6.78E−06 19% +/− 12 62% +/− 11 10% +/− 10 50% +/− 18 Surfactants FattyAcid Amides & C13 Beta-Hydroxy Acid/ 17% 28% 25% Other Surfactants AmideFatty Acid Amides & Castor Oil MEA 3.25E−05 Other Surfactants Fatty AcidAmides & Cocamidopropyl Betaine 25% Other Surfactants Fatty Acid Amides& Coco Hydroxyethyl- 2.84E−07 68% 65% Other Surfactants imidazolineFatty Acid Amides & Cocoamide-MEA (or 11% 13% 34% Other SurfactantsCocoyl Monoethanol- amide) Fatty Acid Amides & Glycerol-PCA-Oleate 41%+/− 6 58% +/− 2 Other Surfactans Fatty Acid Amides & Hexanoamide 20%Other Surfactants Fatty Acid Amides & Hexanoyl Sphingosine 9.99E−05 28%+/− 4 37% +/− 9 Other Surfactants Fatty Acid Amides & Hydroxyethyl-2-3.29E−05 35% 35% Other Surfactants Hydroxy-C12 Amide Fatty Acid Amides &Hydroxyethyl-2- 25% 30% Other Surfactants Hydroxy-C16 Amide Fatty AcidAmides & Lauroyl Sarcosine 20% Other Surfactants Fatty Acid Amides &Lidocaine 12% 0 Other Surfactants Fatty Acid Amides & Linoleamide-DEA(or 59% 12% +/− 3 43% +/− 3 11% +/− 9 51% +/− 15 Other SurfactantsLinoleoyl Diethanolamide) Fatty Acid Amides & Linoleamide-MEA (or1.61E−05 14% 35% 20% +/− 8 35% Other Surfactants Linoleoyl Monoethanol-amide) Fatty Acid Amides & Linoleamidopropyl 69% +/− 18 75% +/− 4 OtherSurfactants Dimethylamine Fatty Acid Amides & Melinamide 64% +/− 15 43%+/− 21 Other Surfactants Fatty Acid Amides & Myristoyl Sarcosine 41% +/−14 11% +/− 11 Other Surfactants Fatty Acid Amides & Oleyl Betaine2.80E−05 47% Other Surfactants Fatty Acid Amides & Palmitamide-MEA  6%23% 12% 33% Other Surfactants Fatty Acid Amides & Stearylhydroxyamide10% 10% Other Surfactants Fatty Acid Amides & Utrecht-1 21% 43% 54% 51%48% +/− 6 Other Surfactants Fatty Acid Amides & Utrecht-2 3.47E−06 42%83% +/− 9 51% 92% +/− 3 Other Surfactants Flavanoids Naringenin 33% 14%Fragrances Allyl Alpha-Ionone 16% +/− 14 22% +/− 23 17% +/− 10 36%/−7Fragrances Alpha-Damascone 3.35E−04 67% +/− 27 83% +/− 12 87% +/− 6 98%+/− 1 Fragrances Alpha-Ionone 9.27E−04 45% +/− 27 49% +/− 30 FragrancesAlpha-Methyl Ionone 67% 77% Fragrances Alpha-Terpineol 26% 25%Fragrances Beta-Damascone 45% 84% 52% 92% Fragrances Brahmanol 70% 75%Fragrances Damascenone 23% 70% 29% 79% Fragrances Delta-Damascone 58%87% 64% 95% Fragrances Dihydro Alpha-Ionone 13% 18% Fragrances EthylSaffranate 51% 49% Fragrances Fenchyl Alcohol 12%  4% FragrancesGamma-Methyl Ionone 21% 38% Fragrances Isobutyl Ionone  8% 45%Fragrances Isocyclogeraniol 18% 16% Fragrances Isodamascone 80% 92%Fragrances Lyral 1.27E−04 76% 71% Fragrances Santalone 23% 12%Fragrances Santanol 15% 43% Fragrances Timberol 34% 33% FragrancesTonalid 50% 33% Fragrances Traseolide 41% 21% Miscellaneous CocoTrimethyl- 27% ammonium Cl- Miscellaneous Urosolic Acid 1.46E−06 21% 28%Noncyclic Citral 20% Fragrances Noncyclic Citronellol 30% 0 FragrancesNoncyclic Farnesol 9.35E−05 23% +/− 18 53% +/− 18 10% +/− 7 53% +/− 19Fragrances Noncyclic Geraniol 7.83E−03 13% 32% Fragrances NoncyclicGeranyl Geraniol 38% +/− 12 81% +/− 6 16% +/− 9 77% +/− 13 FragrancesNoncyclic Linalool 28% 0 Fragrances Noncyclic Nonadieneal 20% FragrancesNoncyclic Pseudoionone 12% 37% Fragrances PhospholipidDioctylphosphatidyl 23% 50% +/− 2 0 17% +/− 17 Ethanolamine UreaDimethyl 22% Imidazolidinone Urea Imidazolidinyl Urea 35%

Retinol Dehydrogenase Activators (B2) Class Compound % Increase RetinolDehydrogenase Phospholipid Phosphatidyl Choline 21% increasePhospholipid Sphingomyelin 26% increase

Retinaldehyde Reductase Inhibitors (B3) Overall % Inhibition ClassCompound TG (IC 50) Retinal Reductase Aldehyde Vanillin 9.70E−03  6%Fatty Acid Arachidic Acid 20% Fatty Acid Arachidonic Acid 49% Fatty AcidLinoleic Acid 1.63E−04 62% +/− 2 Fatty Acid Linolenic Acid 1.34E−04 54%+/− 16 Fatty Acid Myristic Acid 1.72E−05 26% Miscellaneous Amsacrine6.26E−06 22% +/− 8 Miscellaneous Carbenoxolone 3.61E−07 26% +/− 2Miscellaneous Glycyrretinic Acid 8.64E−06 38% +/− 1 PhospholipidPhosphatidyl 37% ethanolamine

CRABPII Antagonists (B4) Overall % Inhibition Class Compound TG(IC 50)CRABPII Fatty Acid Elaidic Acid 6.50E−05 >50% Fatty Acid HexadecanedioicAcid 1.30E−04 >50% Fatty Acid 12-Hydroxystearic Acid 2.91E−05 >50% FattyAcid Isostearic Acid 6.88E−05 >50% Fatty Acids Linseed Oil >50%

Retinoic Acid Oxidation Inhibitors (B5) % Inhibition % InhibitionOverall Retinoic Retinoic Class Compound TG (IC 50) Acid (10 μM) Acid(100 μM) Imidazole Bifonazole 89% 100% Imidazole Climbazole 4.47E−06 80%92% Imidazole Clotrimazole 76% 85% Imidazole Econazole 88% 100%Imidazole Ketoconazole 1.85E−07 84% 84% Imidazole Miconazole 2.78E−0774% 86% Fatty Acid Amides Lauryl Hydroxyethylimidazoline 4.67E−07 &Other Surfactants Fatty Acid Amides Oleyl Hydroxyethylimidazoline3.02E−05 54% 80% & Other Surfactants Flavanoids Quercetin 6.29E−05 40%74% Coumarin Coumarin Quinoline (7H-Benzimidazo [2,1-a]Benz 8.59E−07[de]-Isoquinolin-7-one Quinoline Hydroxyquinoline (Carbostyril) 3.64E−04Quinoline Metyrapone (2-Methyl-1,2-di-3- 47% Pyridyl-1-Propane)

SECTION B Effects of Booster Combinations

In order to assess the effect of combinations of booster molecules anassay is required which encompasses the effect of each of the fivebooster classes. A single enzyme assay is not suitable for this purpose,as it will be specific only for one class of booster molecule. An assaywhich reflects retinoid concentration in keratinocytes is necessary torelate the effects of single booster molecules with combination ofbooster molecules. For this reason, a transglutaminase (Tgase) assay wasutilised. Tgases are calcium dependent enzymes that catalyse theformation of covalent cross-links in proteins. Several Tgase enzymes aremembrane bound in keratinocytes which is important for epidermal cellmaturation. This enzyme is inhibited by retinoic acid. The higher theconcentration of retinoic acid, the greater the inhibition of Tgaseexpression. Hence Tgase is a good marker of both keratinocytedifferentiation and of the retinoid effect on keratinocytes.

Transglutaminase as a Marker of Skin Differentiation

During the process of terminal differentiation in the epidermis, a 15 nmthick layer of protein, known as the cornified envelope (CE) is formedon the inner surface of the cell periphery. The CE is composed ofnumerous distinct proteins which have been cross-linked together by theformation of NΣ-(γ-glutamyl) lysine isodipeptide bonds catalysed by theaction of at least two different transglutaminases (TGases) expressed inthe epidermis. TGase I is expressed in abundance in the differentiatedlayers of the epidermis, especially the granular layer, but is absent inthe undifferentiated basal epidermis. Thus TGase I is a useful marker ofepidermal keratinocyte differentiation with high TGase I levelsindicating a more differentiated state. An ELISA based TGase I assay,using a TGase I antibody, was used to assess the state ofdifferentiation of the cultured keratinocytes in the examples thatfollow.

Keratinocytes (cultured as described above) were plated in 96 wellplates at a density of 4,000-5,000 cells per well in 200 μl media. Afterincubation for two to three days, or until cells are ˜50% confluent, themedia was changed to media containing test compounds (five replicatesper test). The cells were cultured for a further 96 hours after whichtime the media was aspirated and the plates stored at −70° C. Plateswere removed from the freezer, and the cells were washed twice with 200μl of 1×PBS. The cells were incubated for one hour at room temperature(R/T) with TBS/5% BSA (wash buffer, bovine serum albumin). Next theTGase primary antibody was added: 50 μl of monoclonal anti-Tgase I AbB.C. diluted 1:2000 in wash buffer. The primary antibody was incubatedfor 2 hours at 37° C. and then rinsed 6× with wash buffer. Cells werethen incubated with 50 μl of secondary antibody (Fab fragment,peroxidase conjugated anti-mouse IgG obtaining from Amersham) diluted1:4,000 in wash buffer for two hours at 37° C., then rinsed three timeswith wash buffer. Following the rinse with washing buffer, the cellswere rinsed 3× with PBS. For colourimetric development, the cells wereincubated with 100 μl substrate solution (4 mg o-phenylenediamine and3.3 μl 30% H₂O₂ in 10 ml 0.1M citrate buffer pH 5.0) for exactly fiveminutes, R/T, in darkness (under aluminum foil). The reaction wasstopped by the addition of 50 μl 4N H₂SO₄. The absorbance of samples wasread at 492 nm in a 96 well plate UV spectrophotometer. Out of the fivereplicates, four were treated with both antibodies, the fifth one wasuse as a Tgase background control. TGase levels were determined andexpressed as percentage control.

Details of Tgase Assay:

Prior to initiating experiments, to determine the effects ofcombinations of booster molecules standard Tgase assay conditions wereinvestigated. A fully validated Tgase assay was established as follows:

A. Reagents Cells: Human Keratinocytes Neonatal Human foreskin (P2 inT75 flasks; P3 in 96 well assay plates) Primary Antibody: TGm specificBiogenesis (Cat # 5560- monoclonal Ab B.C1 6006) Secondary Ab:Peroxidase Amersham (Cat # NA9310) labeled antimouse Ig F(ab)2 Substratesolution: For 10 ml phosphate citrate buffer 4.0 mg o-phenylenediamineSigma P-7288 3.3 μl of 30% H₂0₂ Sigma H-1909

B. Media/Buffers Keratinocyte Growth Media (KGM) Clonetics (Cat # 3111)Phosphate Buffered Saline; Life Technology Dulbecco's without Ca/MgCl₂)(Cat # 14200-075) Tris Buffered Saline Blocking buffer (1xTBS + 5%BioRad (Cat # 170-6404) dry milk) Washing buffer (1% dry milk in Sigma(Cat # P-7949) TBS + 0.05% Tween 20) Phosphate citrate buffer: 1:1 Sigma(Cat # S-9763) mixture of 0.2M dibasic sodium phosphate and 0.1 M Sigma(Cat # C-1909) citric acid 4 N H₂SO₄

C. Culture ware 96-well polypropylene microtitre Costar (Cat # 3595)plate 96-well polypropylene U-bottom Costar (Cat # 3794) plate T75-ventcap Costar (Cat # 3376)

D: Instrumentation/Equipment Biotek Model EL 340 Microplate Bio-tekInstuments Inc. reader Packard Multiprobe IIE: Cell Culture ProcedureSeeding of Keratinocytes in 96 Well Plates

-   1. A suspension of keratinocytes was prepared at a concentration of    3000 cells/200 μl/well in KGM medium (Used 3×10⁵ cells/12 ml media    in each microtiter plate)-   2. 200 μl of the keratinocyte suspension was transferred into each    of the inner 60 wells only.-   3. 200 μl of KGM media was pipetted into the outer wells (To    maintain thermal equilibrium).-   4. Each plate was incubated at 37° C. and 5% CO₂ for 3 days or until    cells are ˜50% confluent.    Treatment of Keratinocytes with Samples.-   5. Stock solutions of the samples were prepared in DMSO.-   6. The samples were diluted to desired concentration with the final    assay concentration of DMSO being 0.1%.-   7. 20 μl of the sample was transferred into wells and 180 μl of KGM    medium added to give a final assay volume of 200 μl.-   8. Plates were incubated at 37° C. and 5% CO₂ for 72 hours.-   9. Media were completely removed from each well.-   10. Wells were rinsed with 2× with 200 μl of 1×PBS-   11. Finally they were frozen for at least 1.5 hours at −70° C.    F: Transglutaminase Assay-   1. Block:    -   Incubate plates at room temperature with 200 μl/well of blocking        buffer for 1 hour.-   2. Primary Antibody:    -   Aspirate blocking buffer. Incubated with 100 μl/well of        TGm-specific monoclonal antibody B.C1 (diluted 1:2000 in washing        buffer) at 37° C. for at least 2 hours. The primary antibody was        not added in background control wells.-   3. Rinsed wells 6× with washing buffer.-   4. Secondary Antibody:    -   Incubated with 100 μl/well peroxidase labeled anti-mouse        IgF(ab)₂ fragment (diluted 1:4000 in washing buffer) at 37° C.        for 2 hours.-   5. Rinsed wells 3× with washing buffer (added 200 μl) and aspirated    after each rinse.-   6. Rinsed wells 3× with PBS w/o Tween.-   7. Incubated with 100 μl/well substrate solution at room temperature    for exactly 5 minutes.-   8. Stopped reaction with 50 μl/well 4N H₂SO₄.-   9. Read absorbance at 492 nm in the Bio-tek plate reader.    I. Optimization Studies    a. Time Course of Transglutaminase Production

A time course experiment was conducted to determine the optimalincubation time for transglutaminase production in keratinocytes grownin 96-well plates (4000 cells/well). This time course study wasconducted with multiple variables including dose response analyses ofretinoic acid and retinol as well as incubation in the presence of 1.2mM CaCl₂. Although the transglutaminase production in the control cells(0.1% DMSO) was not altered, both retinoic acid and retinol exhibited adose dependent inhibition of transglutaminase production over the fiveday incubation period. The most pronounced retinoid effect was observedon day 2 and day 3. The maximal inhibition was observed on day 2 withthe transglutaminase production being inhibited by 85% and 55% in thepresence of the highest concentration (1 μM) of retinoic acid andretinol respectively. The same experiment was also conducted withvarying cell density (3000 cells/well and 5000 cells/well) andcomparable results were observed.

B: DMSO Sensitivity

Various concentrations of DMSO ranging from 0-2% were tested for theeffect on transglutaminase production in keratinocytes. The assay wassensitive to DMSO concentration with significant inhibition of activity,above 0.5% DMSO. Hence, a final assay concentration of 0.1% was selectedfor subsequent sample concentration studies.

C: Dose Response Curves: Retinoic Acid and Retinol

Based on the data, day 3 was selected as the optimal time and 0.1% DMSOwas selected as the concentration to be used for further testing. Anadditional dose response experiment was carried out with retinoic acidand retinol in the presence of 0.1% DMSO, with the transglutaminaseproduction being assayed on day 3. A good dose response was observed forTgase inhibition by retinoic acid and retinol. 10-7M retinol gave aninhibition of Tgase in the linear range of concentration. Therefore,this concentration of retinol was chosen to evaluate the boostercombinations.

D: Final conditions used to test boosters or combination of boostersDays of incubation of keratinocytes with 3 days retinol and boostersFinal DMSO concentration less than 0.1% Retinol concentration 10-7M (0.1μM) Booster concentrations 10 mM to 0.1 nM

Using the above conditions, dose response for all the different boosters(B1-B5) were tested to identify the best concentration of booster totest in combinations.

Transglutaminase levels were determined and expressed in the Tables B1through B5 either as:

-   (i) % (booster+retinol inhibition/control inhibition)−% (ROH    inhibition/control inhibition), which measures the added effect of    booster+retinol induced TGase inhibition over retinol alone, or-   (ii) as an IC50 value when the inhibitory effect of multiple booster    concentrations was examined—this provides the concentration of    booster which, in combination with a constant retinol concentration    of 10⁻⁷ M, inhibits TGase by 50%.    Booster Combinations and Booster Ratios:

It has been discovered surprisingly that certain compounds increase theendogenous levels of retinoic acid formation from retinol or retinylesters by different mechanisms. These compounds are collectively calledhere as “retinoid boosters”. These include: inhibitors of ARAT/LRAT (B1boosters), inhibitors of retinaldehyde reductase (B3 boosters),inhibitors of retinoic acid binding to CRABP-2 (B4 boosters) andinhibitors of retinoic acid oxidation catalysed by cytochrome P450enzymes (B5 boosters), or certain other compounds which enhance oractivate retinol dehydrogenase (B2 boosters). These boosters are codedas groups B1 through to B5, as seen in chart 1 herein above.

The boosters alone or in combination with each other, potentiate theaction of a retinoid by increasing the amount of retinol available forconversion to retinoic acid and inhibiting the degradation of retinoic,acid. The boosters act in conjunction with a retinoid (e.g. retinol,retinyl ester, retinal, retinoic acid) the latter being presentendogenously in the skin. The preferred compositions, however, include aretinoid in the composition, co-present with a booster, to optimiseperformance.

The present invention includes, in part, a second composition containingfrom about 0.0001% to about 50%, preferably from 0.001% to 10%, mostpreferably from 0.001% to 5% by weight of the composition of at leastone booster compound, or a combination of binary, tertiary, quaternaryor 5 booster combinations. The combined concentration of the boostercombinations of 0.001% to 5% in specified ratios as shown below, inhibittransglutaminase in an in vitro transglutaminase assay to more than 50%,and a cosmetically acceptable vehicle.

The boosters included in the inventive compositions are selected fromthe group consisting of:

-   a. Two boosters, wherein both are selected from the group consisting    of B2, B3 and B4;-   b. Binary combinations of boosters selected from the group    consisting of B1/B2; B1/B3, B1/B4; B1/B5; B2/B3, B2/B4; B2/B5;    B3/B4, B3/B5; B4/B5-   c. Ternary combinations of boosters selected from the group    consisting of B1/B2/B3; B1/B2/B4; B1/B2/B5; B1/B3/B4; B1/B3/B5;    B1/B4/B5; B2/B3/B4; B2/B3/B5; B2/B4/B5; B3/B4/B5-   d. Quaternary combinations of boosters selected from the group    consisting of B1/B2/B3/B4; B1/B2/B3/B5; B1/B2/B4/B5; B1/B3/B4/B5;    B2/B3/B4/B5; and-   e. A combination of five groups of boosters B1/B2/B3/B4/B5.    Booster to Booster Ratios:

The boosters of different classes (B1 to B5) in combinations as shownabove have an optimal concentration of between 0.001% to 5% in acosmetic product at specific ratios as shown below for inhibition ofTgase activity to at least below 50%:

Invention Ratios of boosters to boosters Concentrations Broad 1:10,000to 10,000:1 100 mM to 1 nM  Preferred 1:1000 to 1000:1 10 mM to 10 nMMost preferred 1:100 to 100:1  1 mM to 100 nM Optimum 1:10 to 10:1 0.1mM to 1 μM  Retinoid to Booster Ratios:

The preferred composition includes a retinoid (e.g. retinol, retinylester, and retinaldehyde) in the composition, co-present with a boosteror a combination of the boosters, to optimise performance.

For optimum performance, the concentration of retinoid to booster shouldbe present in the composition in ratios as given below:

Ratios of boosters Invention to retinoids Concentrations Broad 10,000:1to 1:10,000 100 mM-1 nM booster; 0.001-10% retinoids Preferred 1000:1 to1:1000 10 mM-10 nM booster; 0.001-10% retinoid Most 100:1 to 1:100 1mM-100 nM booster; 0.01-1% preferred retinoidConcentrations of Individual Boosters Used in the Examples:

Since the objective is to establish synergistic inhibition oftransglutaminase expression by combinations of the active compounds withretinol, it was essential to determine the dose response profiles (IC₂₀and IC₅₀ values) of the active compounds, when tested individually inthe presence of retinol. The detailed dose response of boostersbelonging to B2-B4 is given in the tables following the IC50 and IC 20table below. This data was used to identify an appropriate sub-maximalinhibitory concentration of each active compound, to eventually make itpossible to identify putative synergistic effects of the mixtures of theactive compounds in the presence of retinol. The data in the followingtable represents the IC₅₀ and IC₂₀ (80% of control) values and theconcentrations used when testing synergies with combinations ofboosters.

In order to demonstrate synergy of two compounds, it is essential toselect concentrations to test that are at most IC20, in other words, acompound concentration that individually boosts the retinol inhibitionof Tgase expression by 20%. Two such compounds should have an additiveinhibition of 40%. Using this strategy to determine concentrationsleaves a window of 40-100% for further inhibition for detecting synergyof the two compounds under examination.

A more challenging concentration criterion would be selectingconcentrations of compounds which alone showed no inhibition effect, butin combination show inhibition. In this study however, we chose an evenmore challenging criteria. We selected concentrations of compounds thatwere 10 to 1000 fold lower than the minimally effective Tgase inhibitingconcentration. Identification of synergistic combinations using suchvery low concentrations would mean that the most effective synergisticcombinations were identified.

Con. Used for Booster synergy (binary, Class Compound Name IC₅₀ IC₂₀tertiary, quaternary) B1 LinoleoylMonoethanolamide 1.61E−05 1.48E−051E−05 to 1E−09 (LAMEA) Palmitamide Monoethanolamide ND ND 1E−06 to 1E−10Oleyl Betaine 2.80E−05 1.08E−05 1E−05 to 1E−8 Naringenin ND ND 1E−05 to1E−09 Echinacea ND ND 1E−05 to 1E−09 Dimethyl imidazolinone ND ND 1E−05to 1E−09 Melinamide ND ND 1E−05 to 1E−09 Geranyl geraniol ND ND 1E−05 to1E−09 Farnesol 9.35E−05 7.82E−05 1E−06 to 1E−09 Geraniol 7.83E−034.72E−03 1E−03 to 1E−07 α-Damascone 3.35E−04 1.69E−04 1E−04 to 1E−08α-Ionone 9.27E−04 1.42E−04 1E−04 to 1E−08 Castor oil Methyl Ester Acid3.25E−05 9.38-E06     1E−06 to 1E−09 (MEA) Ursolic Acid 1.46E−065.94-E07     1E−06 to 1E−09 Utrecht-2 3.47-E06     3.30-E06     1E−06 to1E−09 Cocoyl 2.84E−07 9.21E−08 1E−08 to 1E−11 hydroxyethylimidazolineAcetyl sphingosine (C2 6.78E−06 5.15E−06 1E−06 to 1E−09 Ceramide)Hexanoyl sphingosine (C6 9.99E−05 6.94E−05 1E−05 to 1E−09 Ceramide)Crocetin 3.75E−05 2.52E−05 1E−05 to 1E−09 Lyrial 1.27E−04 4.00E−05 1E−05to 1E−09 N-Hydroxyethyl-2- 3.29E−05 2.40E−05 1E−05 to 1E−09hydroxydodecyl amide B2 Phosphatidyl Choline ND ND 1E−05 to 1E−09Sphingomyelin ND ND 1E−05 to 1E−09 TCC 9.64E−07 6.18-E07     1E−07 to1E−10 1,2-dioctanoyl-sn-glycero-3- ND ND 1E−05 to 1E−09phosphoethanolamide B3 Amsacrine-HCl 6.26E−06 3.30E−06 1E−06 to 1E−09Carbenoxolone 3.61E−07 2.00E−07 1E−07 to 1E−10 Glycyrrhetinic Acid8.64E−06 5.96E−06 1E−06to 1E−09 Linoleic Acid 1.63E−04 8.95E−05 1E−05 to1E−09 Linolenic Acid 1.34E−04 1.21E−04 1E−05 to 1E−09 Arachidonic Acid(Na+ salt) ND ND 1E−05 to 1E−09 Myristic Acid 1.72E−05 1.05E−05 1E−05 to1E−09 Vanilin 9.70E−03 8.47E−03 1E−03 to 1E−06 B4 Hexadecanedioic acid1.30E−04 8.40E−05 1E−05 to 1E−09 12-Hydroxystearic acid 2.91E−051.45E−05 1E−05 to 1E−09 Elaidic acid 6.50E−05 5.88E−05 1E−05 to 1E−09Linseed oil ND ND 1E−05 to 1E−09 Isostearic acid 6.88E−05 6.23E−05 1E−05to 1E−09 2-Hydroxystearic acid ND ND 1E−05 to 1E−09 B5 Climbazole4.47E−06 2.45E−07 1E−07 to 1E−10 Clotrimazole ND ND 1E−05 to 1E−09Miconazole 2.78E−07 8.42E−08 1E−08 to 1E−11 Coumarin ND ND 1E−05 to1E−09 Ketoconazole 1.85E−07 5.52E−08 1E−08 to 1E−11 3,4,-Dihydro-2(1H)-ND ND 1E−05 to 1E−09 quinolinone(Hydrocarbostyril) 2- 3.64E−04 1.70E−041E−04 to 1E−08 Hydroxyquinoline(Carbostyril) Amino Benzotriazole ND ND1E−05 to 1E−09 Lauryl 4.67E−07 2.69E−07 1E−07 to 1E−10hydroxyethylimidazoline Quercetin 6.29E−05 5.11E−05 1E−05 to 1E−09Oleoyl hydroxyethlimidazoline 3.02E−05 5.65E−06 1E−06 to 1E−097H-Benzimidazo[2,1- 8.59E−07 4.69E−07 1E−07 to 1E−09a]Benz[de]-isoquinolin-7-one ND: Not determined or a clear dose responsewas not observed. For synergies, a wide range of concentration (4 ordersof magnitude 10-5 to 10-9M) was tested.Dose Response for Boosters Class B2 to B4

The following tables include the data on the dose response of boostersbelonging to class B2 to B4. Concentration of boosters are given inMolar; mean Tgase level and Standard deviation of 4 replicates isexpressed as % of control (0.1% DMSO and 10-7M retinol). Higher numbers(close to 100 or above 100) indicate no inhibition of Tgase. The lowerthe number, the more potent the inhibitor is at that concentration. TheIC50 and IC20 values were calculated from this dose response table andexpressed in the above table.

B2 Class Boosters

Phosphatidyl choline (B2) Tgase levels Concentration (Mean) Tgase (SD)4.4E−05 90.9 0.01 1.47E−05  120.3 10.6 4.89E−06  70.1 11.4 1.63E−06 98.8 0.00 5.43E−07  86.7 6.19 1.8E−07 75.9 20.5 6.0E−08 87.8 3.9 1.2E−08159 42.3 2.4E−09 85.5 0.39

Sphingomyelin (B2) Tgase levels Concentration (Mean) Tgase (SD)  3.0E−0545 3.21  1.0E−05 77.8 25.5 3.33E−06 76.4 7.55  1.1E−06 98.8 0.003.73E−07 91.6 14.9 1.23E−07 70.0 3.63 4.10E−08 74.6 4.19  8.2E−08 115.21.02 1.65E−09 68.4 2.03 3.29E−10 69.2 2.1

TCC (B2) Tgase levels Concentration (Mean) Tgase (SD) 1.14E−03 36.3 4.6 3.8E−04 3.8 0.96 3.31.23E−04   −3.2 0.91 4.22E−05 −11.2 0 1.41E−06 −.34.88 4.69E−07 15.9 3.52 6.26E−08 18.9 3.12 1.25E−08 100.2 23.3  6.9E−0977.6 21.2  1.0E−09 54.4 11.23

1,2 dioctanoyl-sn-glycero-3-phopshoethanolamide (B2) Tgase levelsConcentration (Mean) Tgase (SD)  1.6E−04 58.1 2.08 5.33E−05 95.4 21.31.78E−05 104 4.01 5.93E−06 129 0.0 1.98E−06 110 8.74 6.58E−07 92.8 15.782.19E−09 88.6 12.3 4.39E−08 127.3 3.39 8.78E−09 119 21.1 1.79E−9  8215.6

B3 Class boosters Amscrine B3 Tgase levels Concentration (Mean) Tgase(SD)  3.0E−05 −10 3.29  1.0E−05 1.8 7.45 3.33E−06 64 4.2  1.1E−06 84 03.73E−07 109 6.2 1.23E−07 65 15.8 4.10E−08 110 10.5  8.2E−08 131 271.65E−09 113 18 3.29E−10 92 8.9

Carbenoxolone (B3) Tgase levels Concentration (Mean) Tgase (SD)  3.0E−06−7.1 0  1.0E−06 27.3 1.15 3.33E−07 51.7 0  1.1E−07 158 0 3.73E−08 1264.67 1.23E−08 81 29 4.10E−09 135 6.88  8.2E−10 112 32 1.65E−10 77.8 10.63.29E−11 64 49

Glyrrhetinic acid (B3) Tgase levels Concentration (Mean) Tgase (SD) 3.0E−04 −0.3 3.9  1.0E−05 0.7 3.55 3.33E−05 2.5 2.1  1.1E−06 96.4 0.003.73E−06 120 33.2 1.23E−07 112 38 4.10E−07 93 11  8.2E−08 225 1081.65E−08 103 11 3.29E−9  100 6.2

Linoleic acid (B3) Tgase levels Concentration (Mean) Tgase (SD)  9.0E−03−6 3.06  3.0E−03 0.1 2.01   1E−03 −16.4 16.3  1.1E−04 4.4 0 3.73E−0479.2 0 1.23E−05 62.6 6.2 4.10E−05 76.8 3.69  8.2E−06 146 44.2 1.65E−07106 20.2 3.29E−07 60.2 2.3

Linolenic acid (B3) Tgase levels Concentration (Mean) Tgase (SD) 9.0E−03 −11 8.7  3.0E−03 −5.7 0.74   1E−03 −7.5 7.8  1.1E−04 −23 03.73E−04 68 0.57 1.23E−05 94.9 17.2 4.10E−05 65.9 0.03  8.2E−06 119 1.61.65E−07 77 8.5 3.29E−07 98 7.0

Myristic acid (B3) Tgase levels Concentration (Mean) Tgase (SD)   1E−03−2 4.1  1.1E−04 −8 2.3 3.73E−04 −6 1.16 1.23E−05 4.10E−05 75.1 1.06 8.2E−06 74.2 10.0 1.65E−07 88.9 8.4 3.29E−07 101 4.47  5.0E−08  1.1E−08

Vanillin (B3) Tgase levels Concentration (Mean) Tgase (SD)  1.4E−02 21.524.2  4.8E−03 93.8 1.7   1E−03 124 15.6  1.1E−04 3.73E−04 101 14.31.23E−05 82 14.6 4.10E−05 98 2.4  8.2E−06 109 22 1.65E−07 80 4 3.29E−0793 41

B4 Class boosters Hexadecanedioic acid (B4) Tgase levels Concentration(Mean) Tgase (SD)   1E−03  1.1E−04 14.2 2.7 3.73E−04 43.4 8.4 1.23E−05130 0 4.10E−05 105 14  8.2E−06 114 12 1.65E−07 95 1.9 3.29E−07  5.0E−0874 6.7  1.1E−08 70 10.4

12-hydroxysteric acid (B4) Tgase levels Concentration (Mean) Tgase (SD)3.73E−04 1.23E−05 −5.2 2.3 4.10E−05 32.4 5.3  8.2E−06 97.6 0 1.65E−0790.2 11 3.29E−07 82 28  5.0E−08 81 3.8  1.1E−08 98 24  2.0E−08 118 28 4.3E−09 71 2.3

Elaidic acid (B4) Tgase levels Concentration (Mean) Tgase (SD)   1E−0312.8 12.1  1.1E−04 8 0.45 3.73E−04 13.8 1.92 1.23E−05 80.9 0 4.10E−0558.2 8.8  8.2E−06 1.65E−07 58 0.13 3.29E−07 69 44  5.0E−08 50.5 3.8 1.1E−08

Linseed Oil (B4) Tgase levels Concentration (Mean) Tgase (SD)   1E−04138 15 3.73E−05 145 2.5 1.23E−05 88 12 4.10E−06 113 0  8.2E−06 113 131.65E−07 96 18 3.29E−07 106 10  5.0E−08 134 22  1.1E−09 83 13  9.9E−1073 15

Isosteric acid (B4) Tgase levels Concentration (Mean) Tgase (SD)   1E−03−8.6 3.4  1.1E−04 1.2 3.0 3.73E−04 −5.3 1.1 1.23E−05 80 00 4.10E−05 677.9  8.2E−06 103 12.3 1.65E−07 95 5.5 3.29E−07 123 0.5  5.0E−08 78 12.2 1.1E−08 78 29

2-hydroxysteric acid (B4) Tgase levels Concentration (Mean) Tgase (SD) 9.1E−04 46.6 6.2 3.73E−04 69.3 8.3 1.23E−04 51 8.8 3.10E−05 96.0 0.0 1.2E−05 105 30 3.65E−06 63 8.0 1.29E−06 80 4.7  2.0E−07 142 34  5.1E−0864 20  1.0E−08 58 17Synergy of Tgase Inhibition with Binary Combinations of Boosters

To investigate synergistic inhibition of Tgase expression bycombinations of 2 different classes of boosters with retinol, selectedcombinations of compounds were tested at concentrations given in theabove table. The concentrations tested were one log order of magnitudeless than the concentration required for minimal inhibition of Tgaseactivity (i.e. IC₂₀). The compounds were tested alone and in combinationand the % inhibition of Tgase is given for each compound and thecombination.

The following examples give the synergistic combinations in all possiblebinary combinations (B1/B2; B1/B3, B1/B4; B1/B5; B2/B3, B2/B4; B2/B5;B3/B4, B3/B5; B4/B5). When the % inhibition of the combination is morethan the inhibition of each compound added together, it indicatessynergy (i.e. Inhibition by combination is greater than inhibition bycompound 1+compound 2). All the binary combination examples given in thefollowing table synergistically inhibited Tgase.

TG as TG as Binary % C % C TG % C combinations Compound 1 Compound 2Compd 1 Compd 2 Combination B1/B2 Dimethyl Phosphatidylcholine 99 97 84imidazolidinone B1/B2 Alpha-demascone Phospahtidylcholine 95 97 86 B1/B2Hexanoyl sphingosine Phospahtidylcholine 109 97 86 B1/B2 Alpha-iononeSphingomyelin 101 98 76 B1/B2 1,2 dioctanoyl-sn- Phosphatidyl choline106 98 78 glycero-3- phosphoethanolamide B1/B2 Alpha-demasconeSphingomyelin 95 84 67 B1/B3 1,2 dioctanoyl-sn- Amsacrine 123 134 75glycero-3- phosphoethanolamide B1/B3 1,2 dioctanoyl-sn- Carbenoxelone123 164 96 glycero-3- phosphoethanolamide B1/B3 Caster oil MEACarbenoxelone 96 164 67 B1/B3 Utrecht-2 Amsacrine 102 98 86 B1/B3Utrecht-2 Carbenoxelone 102 164 91 B1/B3 Hexanoyl sphingosineCarbenoxelone 122 164 78 B1/B3 Lyral Carbenoxelone 120 164 82 B1/B3Castor oil MEA Carbenoxelone 110 164 78 B1/B3 Hexanoyl sphingosineAmsacrine 122 134 92 B1/B3 Hexanoyl sphingosine Eliadic acid 122 144 85B1/B3 Alpha ionone Amsacrine 101 134 78 B1/B3 1,2 dioctanoyl-sn-Glyccyrrhetinic acid 95 92 69 glycero-3- phosphoethanolamide B1/B4Naringenin 2-hydroxy steric acid 95 112 78 B1/B4 Hexanoyl sphingosine2-hydroxy steric acid 99.3 112 77 B1/B4 Lyral Hexadecanoic acid 120 9569 B1/B4 Castor oil MEA Hexadecanedioic acid 110 125 82 B1/B4 Hexanoylsphingosine Isostearic acid 122 146 93 B1/B4 Oleoyl betaineHexadecanedioic acid 99.5 125 80 B1/B5 Hexanoyl sphingosine Cocoyl 99102 68 hydorxyethylimidazoline B1/B5 Farnesol Ketokonazole 98 111 84B1/B5 Hexanoyl sphingosine Miconazole 99 101 56 B1/B5 Hexanoylsphingosine Ketoconazole 99 99 65 B1/B5 Hexanoyl sphingosine Lauryl 9998 51 hydroxyethylimiazoline B1/B5 Utrecht-2 Amino benzotriazole 122 10583 B1/B5 Hexanoyl sphingosine 3,4-dihydro-2 122 102 89 quinolinone B1/B5Hexanoyl sphingosine Amino benzotriazole 122 126 85 B1/B5 Castor oil MEALauryl 110 98 56 hydroxyethylimiazoline B1/B5 Hexanoyl sphingosineClimbazole 122 98 83 B1/B5 Hexanoyl sphingosine Miconazole 122 99 78B1/B5 Hexanoyl sphingosine Ketoconazole 122 110 90 B1/B5 Olecyl beatineketoconazole 96 116 81 B1/B5 Utrecht-2 Lauryl 122 98 57hydroxyethylimiazoline B1/B5 Alpha-demascone Oleoyl 112 73 76hydroxyethylimiazoline B1/B5 Alpha-ionone Lauryl 101 98 49hydroxyethylimiazoline B1/B5 Alpha-ionone Oleoyl 101 73 75hydroxyethylimiazoline B2/B3 Phosphatidyl choline Glycyrrhetinic acid 9892 73 B2/B4 Phosphatidyl choline 2-hydroxy steric acid 98 82 70 B2/B5Phosphatidyl choline Climbazole 98 102 82 B2/B5 Phosphatidyl cholineMiconazole 98 111 92 B2/B5 Phosphatidyl choline Ketoconazole 98 101 89B2/B5 Phosphatidyl choline Lauryl 98 106 82 hydorxyimidazoline B3/B4Amscarine 2-hydroxy steric acid 102 82 75 B3/B4 Myristic acid 2-hydroxysteric acid 110 82 78 B3/B5 Amscarine Aminobenzotriazole 102 98 84 B3/B5Amscarine Dimethyl imidazoline 102 112 94 B3/B5 Myristic acid Climbazole110 102 82 B4/B5 Linseed oil Lauryl hydroxyethyl 98 73 57 imidazolineB4/B5 2-hydroxystearic acid Ketaconazole 92 109 77 B4/B5 Linseed oilOleoyl 98 92 75 hydorxyethylimdazoline B4/B5 2-hydroxystearic acidCoumarin 92 96 70Synergy of Tgase Inhibition with Tertiary Combinations of Boosters

To investigate synergistic inhibition of Tgase expression bycombinations of 3 different classes of boosters with retinol, selectedcombinations of compounds were tested. The concentrations tested wereone log order of magnitude less than the concentration required forminimal inhibition of Tgase activity (i.e. IC₂₀). The compounds weretested alone and in combination and the % inhibition of Tgase is givenfor each compound and the combination. The following examples give thesynergistic combinations in all possible tertiary combinations(B1/B2/B3; B1/B2/B4; B1/B2/B5; B1/B3/B4; B1/B3/B5; B1/B4/B5; B2/B3/B4;B2/B3/B5; B2/B4/B5; B3/B4/B5). The % inhibition of the combination ismore than the inhibition of each compound added together, whichindicates synergy (i.e. Inhibition by combination is greater thaninhibition by compound 1+compound 2+compound 3). All the examples oftertiary combinations of boosters given in the following tablesynergistically inhibited Tgase in the presence of 10-7M retinol

TG as TG as TG as % C TG as % C % C % C Compound 1 Compound 2 Compound 3Compd 1 Compd 2 Compd 3 Combo B1/B2/B3 combinations: PhosphatidylCholine Glycyrrhetinic Castor oil Methyl 88 91 85 53 Acid Ester Acid(MEA) Phosphatidyl Choline Glycyrrhetinic Echinacea 88 91 119 52 AcidPhosphatidyl Choline Glycyrrhetinic Naringenin 88 91 94 52 AcidPhosphatidyl Choline Glycyrrhetinic Acetyl sphingosine 88 91 99 58 Acid(C2 Ceramide) Phosphatidyl Choline Glycyrrhetinic Farnesol 88 91 118 49Acid 1,2-dioctanoyl-sn- Glycyrrhetinic a-Damascone 81 91 89 58glycero-3- Acid phosphoethanolamide 1,2-dioctanoyl-sn- PhosphatidylNaringenin 81 88 94 66 glycero-3- Choline phosphoethanolamide1,2-dioctanoyl-sn- Amsacrine-HCl Linoleoyl 81 79 127 60 glycero-3-Monoethanolamide phosphoethanolamide (LAMEA) 1,2-dioctanoyl-sn-Amsacrine-HCl Palmitamide 81 79 95 63 glycero-3- Monoethanolamidephosphoethanolamide 1,2-dioctanoyl-sn- Glycyrrhetinic a-Damascone 81 9189 58 glycero-3- Acid phosphoethanolamide 1,2-dioctanoyl-sn-Glycyrrhetinic Naringenin 81 91 94 75 glycero-3- Acidphosphoethanolamide 1,2-dioctanoyl-sn- Glycyrrhetinic Echinacea 81 91119 77 glycero-3- Acid phosphoethanolamide 1,2-dioctanoyl-sn-Glycyrrhetinic Dimethyl 81 91 87 67 glycero-3- Acid imidazolinonephosphoethanolamide Castor oil Methyl Carbenoxelone Phosphatidyl 85 9588 63 Ester Acid (MEA) Choline B1/B2/B4 Combinations: B1/B2/B5Combinations: Phosphatidyl Choline Climbazole Echinacea 88 84 119 75Phosphatidyl Choline Climbazole Naringenin 88 84 94 83 PhosphatidylCholine Climbazole Geraniol 88 84 105 76 Phosphatidyl Choline ClimbazoleFarnesol 88 84 118 82 Phosphatidyl Choline Climbazole Acetyl sphingosine88 84 99 82 (C2 Ceramide) Phosphatidyl Choline Miconazole a-Ionone 88 9288 70 Phosphatidyl Choline Miconazole Castor oil Methyl 88 92 85 72Ester Acid (MEA) B1/B3/B4 Combinations: Amsacrine-HCl Dimethyl Elaidicacid 79 87 93 0 imidazolinone □-Ionone Amsacrine-HCl 12-Hydroxystearic68 79 95 62 acid Lyrial Hexadecanedioic Vanillin 97 90 134 81 acidHexanoyl sphingosine Isostearic acid Glycyrrhetinic 104 87 91 58 (C6Ceramide) Acid B1/B3/B5 Combinations: Amsacrine-HCl Dimethyl 2- 79 87 9532 imidazolinone Hydroxyquinoline(Carbostyril) Amsacrine-HCl DimethylLauryl 79 87 52 −13 imidazolinone hydroxyethylimidazoline Amsacrine-HClDimethyl Quercetin 79 87 92 −24 imidazolinone Amsacrine-HCl DimethylOleoyl 79 87 76 39 imidazolinone hydroxyethlimidazoline Amsacrine-HClDimethyl 7H- 79 87 94 32 imidazolinone Benzimidazo[2,1- a]Benz[de]-isoquinolin-7-one Amsacrine-HCl Dimethyl Coumarin 79 87 80 30imidazolinone Hexanoyl Carbenoxolone Oleoyl 104 88 76 64 sphingosine (C6hydroxyethlimidazoline Ceramide) Hexanoyl 3,4,-Dihydro- Vanillin 104 90134 62 sphingosine (C6 2(1H)- Ceramide) quinolinone(Hydrocarbostyril)Amsacrine-HCl Amino Echinacea 79 105 119 48 Benzotriazole Hexanoyl AminoSphingomyelin 104 105 60 69 sphingosine (C6 Benzotriazole Ceramide)Amsacrine-HCl Amino Acetyl sphingosine 79 105 99 −7 Benzotriazole (C2Ceramide) □-Ionone Amsacrine-HCl 7H- 68 79 94 54 Benzimidazo[2,1-a]Benz[de]- isoquinolin-7-one Utrecht-2 Carbenoxolone Quercetin 76 88 9274 Utrecht-2 Carbenoxolone Oleoyl 76 88 76 69 hydroxyethlimidazolineUtrecht-2 Carbenoxolone 7H- 76 88 94 73 Benzimidazo[2,1- a]Benz[de]-isoquinolin-7-one Utrecht-2 Carbenoxolone 3,4,-Dihydro- 76 88 90 702(1H)- quinolinone(Hydrocarbostyril) Myristic Acid Climbazole Geraniol79 84 105 74 Myristic Acid Climbazole □-Damascone 79 84 89 73 MyristicAcid Climbazole Acetyl sphingosine 79 84 99 70 (C2 Ceramide) OleylBetaine Ketoconazole Carbenoxolone 62 85 88 78 Oleyl BetaineKetoconazole Glycyrrhetinic 62 85 91 71 Acid Oleyl Betaine KetoconazoleLinoleic Acid 62 85 11 83 Oleyl Betaine Ketoconazole Linolenic Acid 6285 208 80 Hexanoyl sphingosine 3,4,-Dihydro- Vanillin 104 90 134 62 (C6Ceramide) 2(1H)- quinolinone(Hydrocarbostyril) B1/B4/B5 Combinations:Elaidic acid 2-Hydroxyquinoline Castor oil Methyl 93 95 85 75(Carbostyril) Ester Acid (MEA) Elaidic acid 2-HydroxyquinolineNaringenin 93 95 94 86 (Carbostyril) Elaidic acid 2-Hydroxyquinolinea-Demascone 93 95 89 80 (Carbostyril) Elaidic acid 2-HydroxyquinolineFarnesol 93 95 118 82 (Carbostyril) Elaidic acid 2-HydroxyquinolineCrocetin 93 95 90 78 (Carbostyril) B2/B3/B4 Combinations:1,2-dioctanoyl-sn- Glycyrrhetinic 12-Hydroxystearic 81 91 95 57glycero-3- Acid acid phosphoethanolamide 1,2-dioctanoyl-sn-Glycyrrhetinic Linseed oil 81 91 103 62 glycero-3- Acidphosphoethanolamide 1,2-dioctanoyl-sn- Glycyrrhetinic Elaidic acid 81 9193 75 glycero-3- Acid phosphoethanolamide Phosphatidyl Choline2-Hydroxystearic Arachidonic Acid 88 83 78 60 acid (Na+ salt) B2/B3/B5Combinations: Phosphatidyl Choline Climbazole Linolenic Acid 88 84 20884 Phosphatidyl Choline Climbazole Arachidonic Acid 88 84 78 83 (Na+salt) 1,2-dioctanoyl-sn- Amsacrine-HCl Climbazole 81 79 84 58 glycero-3-phosphoethanolamide 1,2-dioctanoyl-sn- Amsacrine-HCl 7H- 81 79 94 59glycero-3- Benzimidazo[2,1- phosphoethanolamide a]Benz[de]-isoquinolin-7-one 1,2-dioctanoyl-sn- Glycyrrhetinic 3,4,-Dihydro- 81 9190 56 glycero-3- Acid 2(1H)- phosphoethanolamidequinolinone(Hydrocarbostyril) 1,2-dioctanoyl-sn- Glycyrrhetinic 2- 81 9195 75 glycero-3- Acid Hydroxyquinoline(Carbostyril) phosphoethanolamide1,2-dioctanoyl-sn- Glycyrrhetinic Amino 81 91 105 72 glycero-3- AcidBenzotriazole phosphoethanolamide 1,2-dioctanoyl-sn- GlycyrrhetinicLauryl 81 91 52 79 glycero-3- Acid hydroxyethylimidazolinephosphoethanolamide 1,2-dioctanoyl-sn- Glycyrrhetinic Quercetin 81 91 9273 glycero-3- Acid phosphoethanolamide 1,2-dioctanoyl-sn- GlycyrrhetinicClimbazole 81 91 84 54 glycero-3- Acid phosphoethanolamide1,2-dioctanoyl-sn- Glycyrrhetinic Clotrimazole 81 91 79 42 glycero-3-Acid phosphoethanolamide 1,2-dioctanoyl-sn- Glycyrrhetinic Miconazole 8191 82 43 glycero-3- Acid phosphoethanolamide B2/B4/B5 Combinations:Phosphatidyl Choline 2-Hydroxystearic Amino Beozotriazole 88 83 105 77acid Phosphatidyl Choline 2-Hydroxystearic Lauryl 88 83 52 74 acidhydroxyethylimidazoline Phosphatidyl Choline 2-Hydroxystearic Quercetin88 83 92 69 acid Phosphatidyl Choline 2-Hydroxystearic Oleoyl 88 83 7675 acid hydroxyethlimidazoline Phosphatidyl Choline 2-Hydroxystearic7H-Benzimidazo[2,1- 88 83 94 79 acid a]Benz[de]-isoquinolin-7-onePhosphatidyl Choline Climbazole Elaidic acid 88 84 93 81 B3/B4/B5Combinations: Elaidic acid 2-Hydroxyquinoline Carbenoxolone 93 95 88 69(Carbostyril) Elaidic acid 2-Hydroxyquinoline Vanillin 93 95 134 81(Carbostyril) Amsacrine-HCl Amino Linseed oil 79 105 103 45Benzotriazole Myristic Acid Climbazole 12-Hydroxystearic acid 79 84 9581 Myristic Acid Climbazole Linseed oil 79 84 103 81 Elaidic acid2-Hydroxyquinoline Arachidonic Acid (Na+ salt) 93 95 78 63 (Carbostyril)Synergy of Tgase Inhibition with Quaternary Combinations of Boosters

To investigate synergistic inhibition of Tgase expression bycombinations of 4 different classes of boosters with retinol, selectedcombinations of compounds were tested. The concentrations tested wereone log order of magnitude less than the concentration required forminimal inhibition of Tgase activity (i.e. IC₂₀).

The compounds were tested alone and in combination and the % inhibitionof Tgase is given for each compound and the combination. The followingexamples give the synergistic combinations in all possible quaternarycombinations (B1/B2/B3/B4; B1/B2/B3/B5; B1/B2/B4/B5; B1/B3/B4/B5;B2/B3/B4/B5). Synergy was confirmed if the difference in % inhibition ofthe combination (of 4 boosters) is more than 30% that of the inhibitionby 3 booster combinations (i.e. % inhibition of 4 booster combo is equalto or greater than % inhibition of 3 booster combo+30%). All thequaternary combinations of boosters shown in the table given belowshowed synergy.

Tertiary Quarternary (1-3 Difference TG combo; (<30% = Compound 1Compound 2 Compound 3 Compound 4 (% C) TG % C) synergy) B1/B2/B3/B4Combination: Castor oil Methyl Phosphatidyl Glycyrrhetinic 12-Hydroxy-21 64 42 Ester Acid (MEA) Choline Acid stearic acid NaringeninPhosphatidyl Glycyrrhetinic 12-Hydroxy- 15 57 41 Choline Acid stearicacid Linoleoyl 1,2-dioctanoyl- Glycyrrhetinic 12-Hydroxy- −3 40 43Monoethanolamide sn-glycero-3- Acid stearic acid (LAMEA) phosphoethanol-amide Linoleoyl 1,2-dioctanoyl- Glycyrrhetinic Isostearic acid 5 40 35Monoethanolamide sn-glycero-3- Acid (LAMEA) phosphoethanol- amideLinoleoyl 1,2-dioctanoyl- Amsacrine-HCl 12-Hydroxy- −3 42 45Monoethanolamide sn-glycero-3- stearic acid (LAMEA) phosphoethanol-amide Linoleoyl 1,2-dioctanoyl- Amsacrine-HCl Elaidic acid 8 42 34Monoethanolamide sn-glycero-3- (LAMEA) phosphoethanol- amide HexanoylTCC Glycyrrhetinic Isostearic acid 7 54 47 sphingosine (C6 AcidCeramide) Lyrial TCC Vanilin Hexadecanedioic 10 48 38 acid Cocoyl1,2-dioctanoyl- Glycyrrhetinic Isostearic acid 0 37 37hydroxyethylimidazoline sn-glycero-3- Acid phosphoethanol- amide CocoylPhosphatidyl Arachidonic Acid 2-Hydroxy- −1 37 38hydroxyethylimidazoline Choline (Na+ salt) stearic acid Cocoyl1,2-dioctanoyl- Glycyrrhetinic Linseed oil −2 45 47hydroxyethylimidazoline sn-glycero-3- Acid phosphoethanol- amideB1/B2/B3/B5 Combination: Castor oil Methyl Phosphatidyl GlycyrrhetinicClimbazole 20 64 44 Ester Acid (MEA) Choline Acid Castor oil MethylPhosphatidyl Glycyrrhetinic Clotrimazole 26 64 38 Ester Acid (MEA)Choline Acid Castor oil Methyl Phosphatidyl Glycyrrhetinic Miconazole 964 55 Ester Acid (MEA) Choline Acid Castor oil Methyl PhosphatidylGlycyrrhetinic Ketoconazole 5 64 59 Ester Acid (MEA) Choline Acid Castoroil Methyl Phosphatidyl Glycyrrhetinic Lauryl 15 64 49 Ester Acid (MEA)Choline Acid hydroxyethylimidazoline Castor oil Methyl PhosphatidylGlycyrrhetinic Oleoyl 2 64 61 Ester Acid (MEA) Choline Acidhydroxyethlimidazoline Castor oil Methyl Phosphatidyl Glycyrrhetinic7H-Benzimidazo[2,1- 25 64 39 Ester Acid (MEA) Choline Acida]Benz[de]-isoquinolin- 7-one Echinacea Phosphatidyl Glycyrrhetinic12-Hydroxystearic acid 18 62 44 Choline Acid Echinacea PhosphatidylGlycyrrhetinic Climbazole 22 62 40 Choline Acid Echinacea PhosphatidylGlycyrrhetinic Clotrimazole 24 62 38 Choline Acid Echinacea PhosphatidylGlycyrrhetinic Miconazole 13 62 50 Choline Acid Echinacea PhosphatidylGlycyrrhetinic Ketoconazole 12 62 50 Choline Acid Echinacea PhosphatidylGlycyrrhetinic Lauryl 14 62 49 Choline Acid hydroxyethylimidazolineEchinacea Phosphatidyl Glycyrrhetinic Oleoyl 3 62 59 Choline Acidhydroxyethlimidazoline Echinacea Phosphatidyl Glycyrrhetinic7H-Benzimidazo[2,1- 24 62 39 Choline Acid a]Benz[de]-isoquinolin- 7-oneNaringenin Phosphatidyl Glycyrrhetinic Miconazole 1 57 56 Choline AcidNaringenin Phosphatidyl Glycyrrhetinic Ketoconazole 22 57 34 CholineAcid Naringenin Phosphatidyl Glycyrrhetinic Lauryl 10 57 46 Choline Acidhydroxyethylimidazoline Naringenin Phosphatidyl Glycyrrhetinic Oleoyl 257 54 Choline Acid hydroxyethlimidazoline Naringenin PhosphatidylGlycyrrhetinic 7H-Benzimidazo[2,1- 15 57 42 Choline Acida]Benz[de]-isoquinolin- 7-one Palmitamide Phosphatidyl GlycyrrhetinicMiconazole −2 39 41 Monoethanolamide Choline Acid PalmitamidePhosphatidyl Glycyrrhetinic Oleoyl 6 39 33 Monoethanolamide Choline Acidhydroxyethlimidazoline Farnesol Phosphatidyl Glycyrrhetinic Miconazole 343 40 Choline Acid Farnesol Phosphatidyl Glycyrrhetinic Oleoyl 6 43 37Choline Acid hydroxyethlimidazoline Geraniol 1,2-dioctanoyl-Amsacrine-HCl Miconazole 11 47 36 sn-glycero-3- phosphoethanol- amideGeraniol 1,2-dioctanoyl- Amsacrine-HCl Oleoyl 3 47 44 sn-glycero-3-hydroxyethlimidazoline phosphoethanol- amide Linoleoyl 1,2-dioctanoyl-Glycyrrhetinic Climbazole 2 40 37 Monoethanolamide sn-glycero-3- Acid(LAMEA) phosphoethanol- amide Linoleoyl 1,2-dioctanoyl- GlycyrrhetinicMiconazole 5 40 35 Monoethanolamide sn-glycero-3- Acid (LAMEA)phosphoethanol- amide Linoleoyl 1,2-dioctanoyl- GlycyrrhetinicKetoconazole 0 40 40 Monoethanolamide sn-glycero-3- Acid (LAMEA)phosphoethanol- amide Linoleoyl 1,2-dioctanoyl- Glycyrrhetinic Lauryl −240 41 Monoethanolamide sn-glycero-3- Acid hydroxyethylimidazoline(LAMEA) phosphoethanol- amide Linoleoyl 1,2-dioctanoyl- GlycyrrhetinicOleoyl 5 40 35 Monoethanolamide sn-glycero-3- Acidhydroxyethlimidazoline (LAMEA) phosphoethanol- amide Linoleoyl1,2-dioctanoyl- Glycyrrhetinic 7H-Benzimidazo[2,1- 1 40 39Monoethanolamide sn-glycero-3- Acid a]Benz[de]-isoquinolin- (LAMEA)phosphoethanol- 7-one amide Linoleoyl 1,2-dioctanoyl- Amsacrine-HClClimbazole 7 42 35 Monoethanolamide sn-glycero-3- (LAMEA)phosphoethanol- amide Linoleoyl 1,2-dioctanoyl- Amsacrine-HClClotrimazole 10 42 32 Monoethanolamide sn-glycero-3- (LAMEA)phosphoethanol- amide Linoleoyl 1,2-dioctanoyl- Amsacrine-HCl Miconazole5 42 37 Monoethanolamide sn-glycero-3- (LAMEA) phosphoethanol- amideLinoleoyl 1,2-dioctanoyl- Amsacrine-HCl Ketoconazole 11 42 32Monoethanolamide sn-glycero-3- (LAMEA) phosphoethanol- amide Linoleoyl1,2-dioctanoyl- Amsacrine-HCl Lauryl −4 42 46 Monoethanolamidesn-glycero-3- hydroxyethylimidazoline (LAMEA) phosphoethanol- amideLinoleoyl 1,2-dioctanoyl- Amsacrine-HCl Oleoyl 5 42 37 Monoethanolamidesn-glycero-3- hydroxyethlimidazoline (LAMEA) phosphoethanol- amideLinoleoyl 1,2-dioctanoyl- Amsacrine-HCl 7H-Benzimidazo[2,1- 8 42 35Monoethanolamide sn-glycero-3- a]Benz[de]-isoquinolin- (LAMEA)phosphoethanol- 7-one amide Palmitamide 1,2-dioctanoyl- Amsacrine-HClMiconazole 13 43 30 Monoethanolamide sn-glycero-3- phosphoethanol- amidePalmitamide 1,2-dioctanoyl- Amsacrine-HCl Oleoyl 3 43 40Monoethanolamide sn-glycero-3- hydroxyethlimidazoline phosphoethanol-amide Alpha-Damascone 1,2-dioctanoyl- Amsacrine-HCl Miconazole 11 48 37sn-glycero-3- phosphoethanol- amide Alpha-Damascone 1,2-dioctanoyl-Amsacrine-HCl Ketoconazole 13 48 34 sn-glycero-3- phosphoethanol- amideAlpha-Damascone 1,2-dioctanoyl- Amsacrine-HCl Lauryl 15 48 33sn-glycero-3- hydroxyethylimidazoline phosphoethanol- amideAlpha-Damascone 1,2-dioctanoyl- Amsacrine-HCl Oleoylhydroxyethlimidazoline 3 48 45 sn-glycero-3- phosphoethanol- amideCastor oil Methyl Phosphatidyl Carbenoxolone 12-Hydroxystearic acid 3 5552 Ester Acid (MEA) Choline Castor oil Methyl Phosphatidyl CarbenoxoloneClimbazole 6 55 49 Ester Acid (MEA) Choline Castor oil MethylPhosphatidyl Carbenoxolone Miconazole −2 55 57 Ester Acid (MEA) CholineCastor oil Methyl Phosphatidyl Carbenoxolone Ketoconazole 1 55 54 EsterAcid (MEA) Choline Castor oil Methyl Phosphatidyl Carbenoxolone Laurylhydroxyethylimidazoline 4 55 51 Ester Acid (MEA) Choline Castor oilMethyl Phosphatidyl Carbenoxolone Oleoyl 3 55 52 Ester Acid (MEA)Choline hydroxyethlimidazoline Castor oil Methyl PhosphatidylCarbenoxolone 7H-Benzimidazo[2,1- 11 55 44 Ester Acid (MEA) Cholinea]Benz[de]-isoquinolin- 7-one Naringenin Phosphatidyl Linoleic AcidClimbazole −1 45 46 Choline Geraniol Phosphatidyl Linoleic AcidClimbazole 1 40 39 Choline Acetyl sphingosine Phosphatidyl Linoleic AcidClimbazole 0 40 40 (C2 Ceramide) Choline Acetyl sphingosine PhosphatidylLinolenic Acid Climbazole 10 40 30 (C2 Ceramide) Choline Dimethyl TCCAmsacrine-HCl Elaidic acid 14 47 33 imidazolinone Dimethyl TCCAmsacrine-HCl Quercetin 12 44 32 imidazolinone Dimethyl TCCAmsacrine-HCl Coumarin 14 58 44 imidazolinone Hexanoyl TCCGlycyrrhetinic Amino Benzotriazole 8 48 40 sphingosine (C6 AcidCeramide) Alpha-Damascone TCC Myristic Acid Climbazole 10 44 34B1/B2/B4/B5 Combination: Lyrial Vanilin Hexadecanedioic Miconazole 12 4836 acid Lyrial Vanilin Hexadecanedioic Oleoyl 4 48 45 acidhydroxyethlimidazoline Crocetin TCC Elaidic acid 2- 11 48 37Hydroxyquinoline(Carbostyril) Hexanoyl Glycyrrhetinic 12-HydroxystearicAmino Benzotriazole 14 48 33 sphingosine (C6 Acid acid Ceramide)Dimethyl Phosphatidyl 2-Hydroxystearic 7H-Benzimidazo[2,1- 2 44 42imidazolinone Choline acid a]Benz[de]-isoquinolin- 7-one MelinamidePhosphatidyl 2-Hydroxystearic 7H-Benzimidazo[2,1- 5 44 39 Choline acida]Benz[de]-isoquinolin- 7-one Geranyl geraniol Phosphatidyl2-Hydroxystearic 7H-Benzimidazo[2,1- 9 44 35 Choline acida]Benz[de]-isoquinolin- 7-one Cocoyl Phosphatidyl 2-Hydroxystearic7H-Benzimidazo[2,1- −8 44 52 hydroxyethylimidazoline Choline acida]Benz[de]-isoquinolin- 7-one Acetyl sphingosine Phosphatidyl2-Hydroxystearic 7H-Benzimidazo[2,1- 10 44 34 (C2 Ceramide) Choline acida]Benz[de]-isoquinolin- 7-one Crocetin Phosphatidyl 2-Hydroxystearic7H-Benzimidazo[2,1- 10 44 34 Choline acid a]Benz[de]-isoquinolin- 7-oneN,N-Diethyl Phosphatidyl 2-Hydroxystearic 7H-Benzimidazo[2,1- 4 44 40Cocamide(Cocamide Choline acid a]Benz[de]-isoquinolin- DEA) 7-one CocoylPhosphatidyl Elaidic acid Climbazole −4 30 34 hydroxyethylimidazolineCholine B1/B3/B4/B5 Combination: Dimethyl Amsacrine-HCl Elaidic acidMiconazole 7 47 40 imidazolinone Dimethyl Amsacrine-HCl Elaidic acidKetoconazole 6 47 41 imidazolinone Dimethyl Amsacrine-HCl Elaidic acidOleoyl 3 47 44 imidazolinone hydroxyethlimidazoline HexanoylGlycyrrhetinic Isostearic acid Clotrimazole 20 54 34 sphingosine (C6Acid Ceramide) Hexanoyl Glycyrrhetinic Isostearic acid Miconazole 10 5443 sphingosine (C6 Acid Ceramide) Hexanoyl Glycyrrhetinic Isostearicacid Lauryl 20 54 33 sphingosine (C6 Acid hydroxyethylimidazolineCeramide) Hexanoyl Glycyrrhetinic Isostearic acid Oleoyl 5 54 48sphingosine (C6 Acid hydroxyethlimidazoline Ceramide) Crocetin LinoleicAcid Elaidic acid 2-Hydroxyquinoline 0 48 48 (Carbostyril) CrocetinLinolenic Acid Elaidic acid 2-Hydroxyquinoline −2 48 50 (Carbostyril)Castor oil Methyl Linoleic Acid Elaidic acid 2-Hydroxyquinoline −1 31 32Ester Acid (MEA) (Carbostyril) Cocoyl Carbenoxolone Elaidic acid2-Hydroxyquinoline −6 28 34 hydroxyethylimidazoline (Carbostyril)B2/B3/B4/B5 Combination: 1,2-dioctanoyl-sn- Glycyrrhetinic Isostearicacid Ketoconazole 4 37 33 glycero-3- Acid phosphoethanol- amide1,2-dioctanoyl-sn- Glycyrrhetinic Isostearic acid Oleoyl 6 37 31glycero-3- Acid hydroxyethlimidazoline phosphoethanol- amidePhosphatidyl Arachidonic 2-Hydroxystearic Miconazole 6 37 31 CholineAcid (Na+ salt) acid Phosphatidyl Arachidonic 2-Hydroxystearic Oleoyl 537 32 Choline Acid (Na+ salt) acid hydroxyethlimidazoline1,2-dioctanoyl-sn- Glycyrrhetinic Linseed oil Miconazole −1 45 47glycero-3- Acid phosphoethanolamide 1,2-dioctanoyl-sn- GlycyrrhetinicLinseed oil Oleoyl 7 45 38 glycero-3- Acid hydroxyethlimidazolinephosphoethanol- amide Phosphatidyl Carbenoxolone 2-Hydroxystearic7H-Benzimidazo[2,1- 8 44 36 Choline acid a]Benz[de]-isoquinolin- 7-onePhosphatidyl Linoleic Acid 2-Hydroxystearic 7H-Benzimidazo[2,1- −3 44 47Choline acid a]Benz[de]-isoquinolin- 7-one Phosphatidyl GlycyrrhetinicElaidic acid Climbazole −3 30 33 Choline Acid Phosphatidyl Linoleic AcidElaidic acid Climbazole −2 30 32 CholineCosmetically Acceptable Vehicle

The composition according to the invention also comprises a cosmeticallyacceptable vehicle to act as a dilutant, dispersant or carrier for theactive components in the composition, so as to facilitate theirdistribution when the composition is applied to the skin.

Vehicles other than or in addition to water can include liquid or solidemollients, solvents, humectants, thickeners and powders. An especiallypreferred non-aqueous carrier is a polydimethyl siloxane and/or apolydimethyl phenyl siloxane. Silicones of this invention may be thosewith viscosities ranging anywhere from about 10 to 10,000,000centistokes at 25° C. Especially desirable are mixtures of low and highviscosity silicones. These silicones are available from the GeneralElectric Company under trademarks Vicasil, SE and SF and from the DowCorning Company under the 200 and 550 Series. Amounts of silicone whichcan be utilised in the compositions of this invention range anywherefrom 5 to 95%, preferably from 25 to 90% by weight of the composition.

Optional Skin Benefit Materials and Cosmetic Adjuncts

An oil or oily material may be present, together with an emulsifier toprovide either a water-in-oil emulsion or an oil-in-water emulsion,depending largely on the average hydrophilic-lipophilic balance (HLB) ofthe emulsifier employed.

Various types of active ingredients may be present in cosmeticcompositions of the present invention. Various types of activeingredients may be present in cosmetic compositions of the presentinvention. Actives are defined as skin or hair benefit agents other thanemollients and other than ingredients that merely improve the physicalcharacteristics of the composition. Although not limited to thiscategory, general examples include sunscreens, skin lightening agents,and tanning agents.

Sunscreens include those materials commonly employed to blockultraviolet light. Illustrative compounds are the derivatives of PABA,cinnamate and salicylate. For example, octyl methoxycinnamate and2-hydroxy-4-methoxy benzophenone (also known as oxybenzone) can be used.Octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone arecommercially available under the trademarks, Parsol MCX andBenzophenone-3, respectively.

The exact amount of sunscreen employed in the emulsions can varydepending upon the degree of protection desired from the sun's UVradiation.

Another preferred optional ingredient is selected from essential fattyacids (EFAs), i.e., those fatty acids which are essential for the plasmamembrane formation of all cells, in keratinocytes EFA deficiency makescells hyperproliferative. Supplementation of EFA corrects this. EFA'salso enhance lipid biosynthesis of epidermis and provide lipids for thebarrier formation of the epidermis. The essential fatty acids arepreferably chosen from linoleic acid, γ-linolenic acid, homo-γ-linolenicacid, columbinic acid, eicosa-(n-6,9,13)-trienoic acid, arachidonicacid, γ-linolenic acid, timnodonic acid, hexaenoic acid and mixturesthereof.

Emollients are often incorporated into cosmetic compositions of thepresent invention. Levels of such emollients may range from about 0.5%to about 50%, preferably between about 5% and 30% by weight of the totalcomposition. Emollients may be classified under such general chemicalcategories as esters, fatty acids and alcohols, polyols andhydrocarbons.

Esters may be mono- or di-esters. Acceptable examples of fatty di-estersinclude dibutyl adipate, diethyl sebacate, diisopropyl dimerate, anddioctyl succinate. Acceptable branched chain fatty esters include2-ethyl-hexyl myristate, isopropyl stearate and isostearyl palmitate.Acceptable tribasic acid esters include triisopropyl trilinoleate andtrilauryl citrate. Acceptable straight chain fatty esters include laurylpalmitate, myristyl lactate, oleyl eurcate and stearyl oleate. Preferredesters include coco-caprylate/caprate (a blend of coco-caprylate andcoco-caprate), propylene glycol myristyl ether acetate, diisopropyladipate and cetyl octanoate.

Suitable fatty alcohols and acids include those compounds having from 10to 20 carbon atoms. Especially preferred are such compounds such ascetyl, myristyl, palmitic and stearyl alcohols and acids.

Among the polyols which may serve as emollients are linear and branchedchain alkyl polyhydroxyl compounds. For example, propylene glycol,sorbitol and glycerin are preferred. Also useful may be polymericpolyols such as polypropylene glycol and polyethylene glycol. Butyleneand propylene glycol are also especially preferred as penetrationenhancers.

Exemplary hydrocarbons which may serve as emollients are those havinghydrocarbon chains anywhere from 12 to 30 carbon atoms. Specificexamples include mineral oil, petroleum jelly, squalene andisoparaffins.

Another category of functional ingredients within the cosmeticcompositions of the present invention are thickeners. A thickener willusually be present in amounts anywhere from 0.1 to 20% by weight,preferably from about 0.5% to 10% by weight of the composition.Exemplary thickeners are cross-linked polyacrylate materials availableunder the trademark Carbopol from the B.F. Goodrich Company. Gums may beemployed such as xanthan, carrageenan, gelatin, karaya, pectin andlocust beans gum. Under certain circumstances the thickening functionmay be accomplished by a material also serving as a silicone oremollient. For instance, silicone gums in excess of 10 centistokes andesters such as glycerol stearate have dual functionality.

Powders may be incorporated into the cosmetic composition of theinvention. These powders include chalk, talc, Fullers earth, kaolin,starch, smectite clays, chemically modified magnesium aluminum silicate,organically modified montmorillonite clay, hydrated aluminum silicate,fumed silica, aluminum starch octenyl succinate and mixtures thereof.

Other adjunct minor components may also be incorporated into thecosmetic compositions. These ingredients may include coloring agents,opacifiers and perfumes. Amounts of these materials may range anywherefrom 0.001% up to 20% by weight of the composition.

Use of the Composition

The composition according to the invention is intended primarily as aproduct for topical application to human skin, especially as an agentfor conditioning and smoothening the skin, and preventing or reducingthe appearance of wrinkled or aged skin.

In use, a small quantity of the composition, for example from 1 to 5 ml,is applied to exposed areas of the skin, from a suitable container orapplicator and, if necessary, it is then spread over and/or rubbed intothe skin using the hand or fingers or a suitable device.

Product Form and Packaging

The topical skin treatment composition of the invention can beformulated as a lotion, a fluid cream, a cream or a gel. The compositioncan be packaged in a suitable container to suit its viscosity andintended use by the consumer. For example, a lotion or fluid cream canbe packaged in a bottle or a roll-ball applicator, or a capsule, or apropellant-driven aerosol device or a container fitted with a pumpsuitable for finger operation. When the composition is a cream, it cansimply be stored in a non-deformable bottle or squeeze container, suchas a tube or a lidded jar.

The invention accordingly also provides a closed container containing acosmetically acceptable composition as herein defined.

1. A method for treating skin comprising the steps of: (i) applying tothe skin a composition comprising: (a) from 0.01% to 10% of a retinoid;(b) at least one compound that will enhance conversion of retinoid toretinoic acid and prevent the degradation of retinoic acid; (c) acosmetically acceptable vehicle; and (d) fatty alcohol or fatty acid orboth, (e) the compound or compounds that will enhance conversion ofretinoids to retinoic acid and prevent the degradation of retinoic acidcomprise an antimicotic agent; and (ii) treating the skin for wrinkles,psoriasis, age spots or discoloration.
 2. The method according to claim1 wherein the antimicotic agent is bifonazole, climbazole, or a mixturethereof.
 3. The method according to claim 1 wherein the antimicoticagent is climbazole.
 4. The method according to claim 1 wherein the skincare composition further comprises a fatty alcohol and the fatty alcoholis cetyl alcohol.
 5. The method according to claim 1 where the compoundor compounds that will enhance conversion of retinoids to retinoic acidand prevent the degradation of retinoic acid make up from about 0.0001%to 50% of the skin care composition.
 6. The method according to claim 1wherein the compound or compounds that will enhance conversion ofretinoids to retinoic acid and prevent the degradation of retinoic acidmake up form about 0.0001% to 50% of the skin care composition.
 7. Themethod according to claim 1 wherein the compound or compounds willpotentiate the action of retinoids by increasing the conversion ofretinoid to retinoic acid and further wherein the compound or compoundswill cause an action which is an inhibition of ARAT/LRAT activity, anenhancement of retinol dehydrogenase activity, an inhibition of retinalreductase activity, antagonistic to CRABP-II binding of retinoic acidand/or an inhibition of cytochrome P450 dependent retinoic acidoxidation.
 8. The method according to claim 1 wherein the compositioncomprises climbazole and cetyl alcohol.
 9. The method according to claim1 wherein the composition comprises bifonazole and cetyl alcohol. 10.The method according to claim 1 wherein the composition comprisesbifonazole, climbazole and cetyl alcohol.
 11. The method according toclaim 1 wherein the composition further comprises a sunscreen and skinlightening agent.
 12. The method according to claim 2 wherein thecomposition further comprises linoleoyl monoethanolamide, palmitamidemonoethanolamide, castor oil methyl ester acid or a mixture thereof. 13.The method according to claim 5 wherein the compound or compounds thatwill enhance conversion of retinoids to retinoic acid and prevent thedegradation of retinoic acid make up from about 0.001% to 10% of theskin care composition.
 14. The method according to claim 6 wherein thecompound or compounds that will enhance conversion of retinoids toretinoic acid and prevent the degradation of retinoic acid make up fromabout 0.001% to 10% of the skin care composition.